Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

High coenzyme affinity chimeric amine dehydrogenase based on domain engineering

High coenzyme affinity chimeric amine dehydrogenase based on domain engineering *Correspondence: xqmu@jiangnan.edu.cn Jialin Li and Xiaoqing Mu contributed equally Laboratory of Brewing Microbiology and Applied Enzymology, School of Biotechnology, Jiangnan University, Wuxi 214122, China Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Li et al. Bioresources and Bioprocessing (2022) 9:33 Page 2 of 10 140 to the terminus 366 by the L-AmDH from Bacil- Introduction lus stereothermophilus) via domain shuffling, which can Chiral amines are an important class of chiral building catalyze adamantyl methyl ketone to adamantyl ethyl- blocks that are widely used in fine chemicals, agriculture, amine unlike parent proteins and strongly improve ther- biologically active natural products, and pharmaceuti- mal activity. Ch1-AmDH (a chimeric enzyme obtained cal intermediates (Jiang and Fang 2020; Wang and Reetz through domain shuffling of first-generation variants) 2015). Compared with most chemical synthesis path- was used to understand the catalytic mechanism and ways that require harsh reaction conditions such as high the molecular discriminants that are crucial for the effi - temperature and high pressure, enzyme catalysis synthe- cient catalytic activity of AmDHs in 2019 (Tseliou et  al. sizes chiral chemicals through its high stereo selectivity, 2019). Crystal structure studies then on AmDHs sug- mild reaction conditions, and environmental friendli- gested that the monomer structure of AmDH consists of ness (Abrahamson et  al. 2012; Chen et  al. 2018; Sharma two independent substrate-binding and cofactor-binding et al. 2017). Among the enzymes that have been reported domains (Son et  al. 2015a). Subsequently, a chimeric for the synthesis of chiral amines, AmDH only requires enzyme that changes the specificity of the coenzyme has cheap ammonium ions as the amino donor, with water as been reported. By replacing the cofactor NA D bind- its only by-product, considered a greener synthetic route ing domain from Clostridium symbiotic to the cofactor (Chen et al. 2015; Huang et al. 2016). NADP binding domain of glutamate dehydrogenase At present, there are two main types of AmDH: natu- from Escherichia coli, the coenzyme dependency was ral AmDH and engineered AmDH modified from natural + + changed from NADP to NAD (Sharkey and Engel, amino acid dehydrogenase (Tseliou et  al. 2020). How- 2009). This chimeric enzyme showed NAD dependence ever, natural AmDH has few origins, a narrow substrate and high catalytic efficiency. This strategy suggests that spectrum, and low catalytic activity, greatly limiting their the combination or substitution of enzyme domains may application in the synthesis of chiral amines (Mayol et al. contribute to the overall property changes. 2016). Since its creation by Abrahamson et  al. (2012) in Recently, phenylalanine amine dehydrogenases that 2012, worldwide attention has focused on improving the catalyze difficult aromatic ketone substrates, important substrate spectrum and increasing the catalytic efficiency precursors of pharmaceutical intermediates, have been by rationally designing a directed evolution-engineered gradually reported (Abrahamson et  al. 2013; Ruffoni AmDH. Most of the research was aimed at modifying et  al. 2019; Zoi et  al. 2017). However, their lower coen- the site near the substrate-binding pocket to improve the zyme affinity is limited in industrial production (Kataoka properties of AmDH (Ducrot et  al. 2020; Grogan 2018; and Tanizawa 2003; Li et al. 2014; Zhu et al. 2016). Here, Itoh et al. 2000; Wu et al. 2021). As these are coenzyme- We report a chimeric enzyme, cFLF-AmDH, based on dependent enzymes, reaction efficiency can be improved homologous sequence alignment and structural analysis using a higher coenzyme concentration or increasing of the independent substrate and coenzyme of AmDH. the affinity between the enzyme and coenzyme (Frank - The coenzyme-binding domain of L-AmDH from Bacil - lin et  al. 2020; Itoh et  al. 2000; Zhou et  al. 2019). The lus cereus was used to replace the corresponding region latter improves the reaction efficiency and reduces the of F-AmDH from Bacillus badius, which has a lower dosage of expensive coenzymes to reduce production coenzyme affinity. The constructed cFLF-AmDH had costs. This strategy is gradually being applied (Cai et  al. high coenzyme affinity and catalytic efficiency, and fur - 2020; Jiang and Wang 2020; Li et  al. 2019; Wang et  al. ther broadened the substrate spectrum based on inherit- 2015). In 2021, by alignment with the coenzyme-binding ing the substrate specificity of F-BbAmDH. Subsequently, domain sequence of amino acid dehydrogenase which we used molecular dynamics (MD) simulations to clarify has higher coenzyme affinity, Meng et al. (2021) adjusted the factors that help to increase the coenzyme affinity the coenzyme-binding cavity increasing the NADH activ- and catalytic efficiency observed in kinetic analysis and ity of leucine dehydrogenase (LaLeuDH) from Labren- conversion experiments. This enables the rational design zia aggregata by introducing a double mutation in the of coenzyme-binding domains to screen good candidates coenzyme-binding region, thereby increasing reaction for improved catalytic efficiency and cofactor affinity. efficiency. With the continuous deepening of research, the modi- Materials and methods fication of AmDH is not limited to directed evolution. Strains, plasmids, and chemicals Domain shuffling provides an alternative method for F-BbAmDH from Bacillus badius、L-BcAmDH from obtaining enzymes with improved properties (Kataoka Bacillus cereus and L-EsAmDH from Exiguobacte- et  al. 1994). In 2014, Bommarius et  al. (2014) created a rium sibiricum were generated in the laboratory. Glu- novel chimeric amine dehydrogenase (residues 1–149 cose dehydrogenase from Bacillus amyloliquefaciens were contributed by F-AmDH from Bacillus badius and Li  et al. Bioresources and Bioprocessing (2022) 9:33 Page 3 of 10 (BaGluDH) was purchased from Sigma-Aldrich Corp. Kinetic parameter determination (Beijing, China). Escherichia coli BL21 (DE3) and plas- Kinetic parameters of parental F-BbAmDH and cFLF- mid pET-28a (+) were purchased from Novagen (Nan- AmDH were determined in N H Cl/NH OH buffer 4 4 jing, China) as the gene expression host and vector, (100 mM, pH 8.5) at 30 °C in different concentrations of respectively. p-Fluorophenyl acetone (p-FPA) was bought p-FPA (with concentration range from 0.05 to 50 mM) or from J&K Co. Ltd. (Shanghai, China). Isopropyl-β-D- NADH (with concentration range from 0.01 to 0.5 mM). + −1 −1 thiogalactoside (IPTG), kanamycin, NADH, and NAD The kinetic constant (K , mmol·L ; k, min ) and m cat −1 −1 −1 were from TCI (Shanghai, China). All other chemicals catalytic efficiency (k /K, L ·min ·mmol ) were cat m were analytical grade and commercially available. calculated using a nonlinear curve fitting of initial veloc - ity versus substrate concentration data to the Michaelis– Construction of cFLF‑AmDH expression vectors Menten equation by Origin software. All the values were The coenzyme-binding domains of two amine dehydro - averaged from three replicates with standard deviations. genases were predicted using the NCBI (https:// www. ncbi. nlm. nih. gov). Chimeric amine dehydrogenase Enzyme activity assay (cFLF-AmDH) was introduced using homologous recom- Enzyme activity was determined by monitoring the bination technology containing the coenzyme-binding change in absorbance at 340  nm at 30  °C using a Multi- domain of L-BcAmDH and the substrate-binding domain Skan GO UV-spectrometer (Thermo Fisher Scientific), of F-BbAmDH. The corresponding primers were syn - which corresponds to the change in the concentration thesized by Shenggong Bioengineering (Shanghai) Co., of NADH (Li et  al. 2014). For reductive amination, the Ltd. (Shanghai China). The PCR program was run for reaction mixture (200 μL) contained 20  mM substrate, 30 cycles under the following conditions: 30  s at 98  °C, 0.2  mM NADH, 2  M NH Cl/NH OH buffer (pH 9.0) 4 4 10  s at 98  °C, 30  s at 55  °C, 70  s at 72  °C, and 10  min at and a certain amount of purified enzyme. Enzyme activ - 72  °C, after which it was kept at 10  °C. The constructed ity unit (U) was defined as the amount of enzyme which plasmids were then isolated and sequenced prior to their catalyzes the production (or consumption) of 1  µmol of introduction into E. coli BL21 (DE3). The chimeric strain NADH per min under the above conditions. E. coli BL21/pET-28a-cFLFAmDH was obtained after confirmed by DNA sequencing. Enzymatic properties determination The enzyme activity of F-BbAmDH and cFLF-AmDH Expression and purification of enzymes was measured at various pH (7.0–11.0) in NH Cl/ Phenylalanine amine dehydrogenase (F-BbAmDH) NH OH buffer (30 °C). The pH stability was determined was cultured in Luria–Bertani (LB) medium contain- by enzymatic incubation in N H Cl/NH OH buffer at dif - 4 4 −1 ing kanamycin (50  mg·L ) at 37  °C and with shaking ferent pH (7.0–11.0) at 30 °C for 2 h. at 200  rpm for 2  h. Protein expression was induced by The enzyme activity was measured and calculated at the addition of IPTG at a final concentration of 0.2  mM various temperatures (30–70 °C) in 2 M NH Cl/NH OH 4 4 when the bacteria reached an OD value of 0.6–0.8, buffer (pH 9.0). Thermal stability of F-BbAmDH and and the cells were then cultured at 17  °C and 200  rpm cFLF-AmDH was characterized by half-life. The half-life for 12 h. cFLF-AmDH was cultured in an auto-induction (t ) was calculated by incubating in 2 M NH Cl/NH OH 1/2 4 4 medium at 37 °C with shaking at 200 rpm for 2 h. When buffer (pH 9.0) at 55  °C for different time. The t value 1/2 the OD value of culture was 0.6–0.8, the temperature at 55  °C was calculated using the following formula: −1 was adjusted to 17  °C to induce protein expression and t = ln2 k (k is the first-order rate constant, which is 1/2 cultured for 60  h. The culture was collected by centrifu - derived from the semi-log plot of incubation time and gation at 4  °C and 8000  rpm for 5  min and then stored residual activity) (Le et  al. 2012). Specific activity before at −  80  °C until further use. Proteins were expressed in incubation was normalized as 100%. E. coli BL21 (DE3) with His -tag at the C-terminus. Cells were lysed by ultrasonic cell crusher and the supernatant Biotransformation and analytical methods was collected by centrifugation at 12,000 × g for 30  min The reaction mixture (10  ml) consisted of 2  M NH Cl/ at 4 °C. Proteins were purified with an ÄKTA purifier sys - NH OH buffer (pH 8.5), 20  mM NAD , 2  mM P-FBA tem (GE Healthcare, Little Chalfont, UK) using Ni–NTA and 20 μg protein. The reaction was carried out at 37 °C affinity columns and Superdex 200 chromatography. The with shaking at 200  rpm for 24  h. Samples (1  mL) were enzyme purity was determined by SDS-PAGE. drawn at intervals and kept in boiling water for 10  min Li et al. Bioresources and Bioprocessing (2022) 9:33 Page 4 of 10 to terminate the reaction. The reaction liquid sample Results and discussion (1.0 ml) was extracted twice with 1.0 ml ethyl acetate, and Sequence and structure analysis of parent AmDHs 1.0 ml extract was taken for product determination. The F-BbAmDH from Bacillus badius (Abrahamson et  al. solutions were then passed through a 0.22-μm filter. 2013) is an engineered amine dehydrogenase that cata- The conversion of amine products were performed with lyzes aliphatic and aromatic ketone substrates. Compared GC-FID analysis on a 7890B GC (Agilent) using nitrogen with other reported engineered amine dehydrogenases, as the carrier gas. Analytic conditions: Grace Econo-Cap the natural affinity for coenzymes of F-BbAmDH is low, EC-WAX + column (30  m × 0.25  mm × 0.25  μm). Split increasing the cost of industrial production. Based on ratio 5:1, pressure 120.0  kPa. Column temperature pro- domain recombination technology, coenzyme domain −1 gram: starting at 90 °C, hold for 2 min, with 10 °C·min replacement is an effective means of improving parental to 180 °C, hold for 2 min. coenzyme affinity. Two high-affinity coenzyme domain donors (Table  1), Structure modeling, molecular docking, and MD L-BcAmDH from Bacillus cereus (Mu et  al. 2021) and simulation L-EsAmDH from Exiguobacterium sibiricum (Chen et  al. Amino acid sequence alignment was performed using the 2018), were used as donors to obtain a structure similar to MUSCLE server (https:// www. ebi. ac. uk/ Tools/ msa/ mus- that of the amino acid dehydrogenase superfamily (Li et  al. cle/) (Madeira et al. 2019) and displayed using the Esprit 2014). All  three  enzymes  were  derived  from the  introduc- server (https:// espri pt. ibcp. fr/ ESPri pt/ ESPri pt/) (Rob- tion to KS/NL double mutations that alter the substrate spec- ert and Gouet 2014). Protein structure was predicted ificity at the catalytically  active  center of  the corresponding by Robetta server (https:// robet ta. baker lab. org/). Dock- amino  acid  dehydrogenases. To investigate the structural ing of proteins to ligands was obtained with Auto Dock homology of F-BbAmDH, L-BcAmDH, and L-EsAmDH, Tools (http:// autod ock. scrip ps. edu/ resou rces/ adt). Pro- the amino acid sequences, and tertiary structures were tein structure maps were produced by the 3D visualiza- compared using sequence alignment and Robetta server tion software Pymol (https:// www. pymol. org). Molecular modeling. The whole length of F-BbAmDH had 46.84% and dynamics (MD) simulations using GROMACS version 49.48% amino acid sequence homology with L-BcAmDH 5.0.2 and AMBER force field (Ganjoo et al. 2020). System and L-EsAmDH, respectively (Additional file  1: Figure S1). was built in a three-centered water model in an orthog- Meanwhile, F-BbAmDH shared 47.84% and 48.78% amino onal box that extends 10  Å from the dissolved atoms in acid sequence homology with L-BcAmDH and L-EsAmDH all three dimensions in order to create buffers between in the coenzyme-binding region, respectively. The three them. Use the steepest descent algorithm for energy enzymes were highly similar in their tertiary structure (Addi- minimization, a total of 10,000 steps (Son et  al. 2015b). tional file  1: Figure S2). L-BcAmDH was chosen as a cofactor In addition, the kinetics of the protein–coenzyme com- domain donor for the higher coenzyme affinity of approxi - plex and the protein–ligand complex were simulated for mately 2.5-fold compared with L-EsAmDH (Table 1). 50 ns and the system was heated to 300 K at a pressure of 1.01 bar (Ganjoo et al. 2020). Molecular dynamics simu- lation trajectories for binding free energy calculations Coenzyme affinity of chimeric amine dehydrogenase were performed using the MM/GBSA method, defined The structural study suggested that F-BbAmDH is ΔG = G -G -G (Genheden and Ryde 2015). composed of a substrate-binding domain, coenzyme- bind PL P L binding domain, and terminal structure. The chimeric Table 1 Kinetic parameters of the parent for substrate NADH Table 2 Kinetic parameters of chimeric amine dehydrogenase and F-BbAmDH for substrate NADH −1 Enzyme Substrate K (mM) k (min ) k /K m cat cat m −1 −1 (mM  min ) −1 Enzyme Substrate K (mM) k (min ) k /K m cat cat m −1 −1 (mM  min ) F-BbAmDH NADH 0.16 ± 0.02 0.65 ± 0.01 4.03 L-BcAmDH NADH 0.021 ± 0.004 1.09 ± 0.1 51.40 F-BbAmDH NADH 0.16 ± 0.02 0.65 ± 0.01 4.03 cFLFAmDH NADH 0.045 ± 0.005 1.42 ± 0.22 31.56 L-EsAmDH NADH 0.052 ± 0.005 1.62 ± 0.22 31.15 Determination of coenzyme kinetic parameters using the same substrate Comparison of coenzyme kinetics of cFLF-AmDH and F-BbAmDH. Determination 2-pentanone. The values were generated by fitting the initial specific activity of coenzyme kinetic parameters using the same substrate 2-pentanone. data to the Michaelis–Menten equation using nonlinear regression with The values were generated by fitting the initial specific activity data to the GraphPad Prism software. Value is means ± standard deviations. All reactions Michaelis–Menten equation using nonlinear regression with GraphPad Prism involved in the kinetic constant calculations were analyzed using a 2 M NH Cl/ software. At the same substrate concentration, the kinetic parameters of NH OH buffer at optimum pH and temperature. All experiments were repeated the coenzyme are measured using NADH of different concentrations, that is, 3 times 0.05–0.5 mM Li  et al. Bioresources and Bioprocessing (2022) 9:33 Page 5 of 10 amine dehydrogenase cFLF-AmDH was built from the stabilities were inversely proportional. The RMSD value substrate-binding domain and terminal structure from of the cFLF-AmDH complex was between F-BbAmDH F-BbAmDH and the coenzyme-binding domain from and L-BcAmDH, which was aligned with the measured L-BcAmDH. It was soluble (Additional file  1: Figure catalytic properties. The cFLF-AmDH did not fully char - S3), expressed by self-induction culture, and the kinetic acterize the full activity of L-BcAmDH. However, com- parameters of the coenzyme were determined accord- pared with F-BbAmDH, there was a significant change. ing to the Michaelis–Menten equation using nonlinear regression with GraphPad Prism software. Compared Enzymatic properties of cFLF‑AmDH with the F-BbAmDH, the k /K values for NADH of According to the results of the coenzyme affinity analy - cat m cFLF-AmDH increased by 4.2-fold. The k values were sis and MD simulation, the chimeric enzyme had better cat 2.2-fold higher and the K values were twofold lower properties. Subsequently, we investigated the enzymatic (Table 2). These results indicated that cFLF-AmDH had properties of cFLF-AmDH and F-BbAmDH. The optimal a stronger affinity and catalytic efficiency for NADH. reaction temperatures of cFLF-AmDH and F-BbAmDH were 60  °C and 55  °C, respectively (Fig.  2a). However, MD simulation of chimeric amine dehydrogenase‑NADH thermal optima of cFL1-AmDH and cFL2-AmDH (Bom- complexes marius et  al. 2014) were both greater than 70  °C. The To understand the molecular mechanism of the cata- thermal stabilities researched were carried out at 55  °C lytic efficiency improvement of cFLF-AmDH, all-atom by calculating the half-life (t ). The half-life of cFLF- 1/2 MD simulations for F-BbAmDH, L-BcAmDH, and AmDH was 9.6  h which was 160% higher than that of cFLF-AmDH as well as their complexes with NADH at parent F-BbAmDH (6.0 h; Fig. 2b and 2e) and the half-life 300  K were performed to analyze the structural changes of cFL1-AmDH (Bommarius et al. 2014) also greater than that occur in proteins. The root-mean-square deviation 500 min at 55 °C. (RMSD) was used to measure the average deviation of the The optimal reaction pH value and stability of protein conformation from the original structure (Hynd- F-BbAmDH and cFLF-AmDH were similar (Fig.  2c and man and Koehler 2006). As shown in Fig.  1, the RMSD 2d). Although the optimal reaction pH value was 10.0, evolution of the complex of F-BbAmDH and NADH the residual activity after 2  h of incubation decreased as showed that the variation range was 2.5–3.5  Å  under the pH value increased. It retained more than 80% and Cα, 3.0–4.5 Å under the side chain, and 4.0–5.5 Å under only 20–25% activity at a neutral pH (7.0) and alkaline the heavy atom. The RMSD evolution of the L-Bc AmDH pH (10.0), respectively. and NADH complexes also showed that Cα, side chains, heavy atoms varied between 1.5–2.0  Å, 2.0–2.5  Å, and Substrate specificity of cFLF‑AmDH 2.5–3.5  Å, respectively. However, the trajectory of the Structurally, part of the amino acid residues in the coen- complex of cFLF-AmDH and NADH changed in the zyme-binding domain will participate in the formation of range of 1.5–2.5  Å under Cα, 2.5–3.5  Å under the side the substrate-binding pocket, so the replacement of the chain and 3.0–4.5 Å under heavy atoms. For composites coenzyme-binding domain may cause changes in sub- with similar structures, the RMSD values and structural strate specificity and activity. The activities of a series Fig. 1 Comparison and analysis of differences between F-BbAmDH, L-BcAmDH and cFLF-AmDH based on MD simulation. The above plot shows the RMSD evolution of F-BbAmDH (a), L-BcAmDH (b) and cFLF-AmDH (c) during the 50-ns simulation at 300 K and 1.01 bar pressure. The first frame is used as the reference. All protein frames are first aligned on the reference frame backbone, and then the RMSD of Cα (black), side chain (red), and heavy atoms (blue) were calculated Li et al. Bioresources and Bioprocessing (2022) 9:33 Page 6 of 10 Fig. 2 Comparison of enzymatic properties of cFLF-AmDH and F-BbAmDH. a Optimum reaction temperature. b Temperature stability. c Optimum reaction pH. d pH stability. e Half-life. The error bars showed the standard deviations of three replicates Li  et al. Bioresources and Bioprocessing (2022) 9:33 Page 7 of 10 −1 Table 3 Specific activity (mU mg ) of F-BbAmDH and cFLF- higher catalytic activity and broad substrate specificity AmDH toward aliphatic ketones than F-BbAmDH. The activity of cFLF-AmDH to 2-pen - −1 a tanone, p-fluorophenyl acetone (p-FPA), 4-methylpro - Substrates Specific activity (mU mg ) piophenone, and p-methoxypropiophenone increased by F‑BbAmDH cFLF‑ AmDH 500%, 250%, 200%, and 170%, respectively. cFLF-AmDH accepted new substrates such as 4-methyl-2-pentanone, 4-Methyl-2-butanone n.a. n.a 5-hydroxy-2-pentanone, acetophenone, and 3-methy- 4-Hydroxy-2-butanone n.a n.a lacetophenone. Our cFLF-AmDH had activity against 2-Pentanone 33.5 143.32 acetophenone and pFPA at 30 °C, however, cFL1-AmDH 4-Methyl-2-pentanone n.a 25.29 was hardly active at 30 °C (Bommarius et al. 2014). 5-Hydroxy-2-pentanone n.a 34.75 p-FPA was selected as the test substrate because pheny- Activity was measured in NH Cl/NH OH buffer (2 M, pH 9.0) containing 0.2 mM 4 4 lacetone, the simplest ketone analog of phenylpyruvate, NADH and 20 mM substrate at 30 °C n.a. = no measurable activity the natural substance of phenylalanine dehydrogenase, was not readily available as a controlled substance and its vitality increased the most in this study. To observe −1 Table 4 Specific activity (mU mg ) of F-BbAmDH and cFLF- the changes in coenzyme affinity and substrate affinity, AmDH toward aromatic ketones the kinetic parameters were determined with pFPA and −1 a Substrates Specific activity (mU mg ) NADH at various concentrations. The obtained kinetic data are summarized in Table  5. cFLF-AmDH yielded F‑BbAmDH cFLF‑ AmDH 1.7-fold lower K values and 1.2-fold increases in k val- m cat Acetophenone n.a. 21.96 ues. This led to a 2.1-fold increase in k /K values for cat m 2-Methylacetophenone n.a n.a NADH. Compared with F-BbAmDH, cFLF-AmDH gave 3-Methylacetophenone n.a 23.32 threefold higher k values and 2.6-fold higher K val- cat m 4-Methylacetophenone n.a n.a ues for pFPA reduction, resulting in a 1.2-fold increase in 4-Fluoroacetophenone n.a n.a the k /K values. These results indicated that the affin - cat m 3-Hydroxyacetophenone n.a n.a ity of cFLF-AmDH for coenzymes increased and that the 4-Methoxyacetophenone n.a n.a catalytic efficiency of the coenzyme and substrate pFPA 4-Fluorophenylacetone 719.8 1759.4 improved. For comparison with cFL1-AmDH and cFL2- 4-Methylpropiophenone 377.9 838.1 AmDH, we also measured coenzyme kinetic param- 4-Hydroxypropiophenone n.a n.a eters at 60  °C. The K and K values of cFLF-AmDH m cat −1 P-Methoxyphenylacetone 199.1 337.9 were 0.05  mM and 132.44  min , respectively, at 60  °C. 4-(4-Methoxyphenyl)-2-butanone n.a n.a Although the K value was not much different, the K m cat Activity was measured in NH Cl/NH OH buffer (2 M, pH 9.0) containing 0.2 mM value of cFLF-AmDH was 2.5-fold larger than that of 4 4 NADH and 20 mM substrate at 30 °C cFL1-AmDH (Bommarius et al. 2014). n.a. = no measurable activity In the docking analysis, the distance between p-FPA and the side chain of cFLF-AmDH-K90 was 2.0  Å. The K90 was the key residue for the interaction between of aliphatic ketone substrates and aromatic ketone sub- ammonia and the substrate (Fig.  3). MD simulation strates of cFLF-AmDH and F-BbAmDH were investi- studies the structural changes that occur when a sub- gated and compared. For both aliphatic (Table  3) and strate binds to a protein (Fig.  4). The root-mean-square aromatic (Table  4) ketone substrates, cFLF-AmDH had Table 5 Kinetic parameters of the F-BbAmDH and cFLF-AmDH Enzyme pFPA NADH −1 −1 K (mM) k (min ) k /K K (mM) k (min ) k /K m cat cat m m cat cat m −1 −1 −1 −1 (min  mM ) (min  mM ) F-BbAmDH 8.262 ± 1.13 56.74 ± 4.83 6.60 0.86 ± 0.05 47.66 ± 3.46 55.40 cFLF-AmDH 21.81 ± 2.67 173.4 ± 6.94 7.95 0.49 ± 0.02 56.91 ± 5.12 116.14 The values were generated by fitting the initial specific activity data to the Michaelis–Menten equation using nonlinear regression with GraphPad Prism software. Value is means ± standard deviations. All reactions involved in the kinetic constant calculations were analyzed using a 2 M NH Cl/NH OH buffer at optimum pH and 4 4 temperature. All experiments were repeated 3 times Li et al. Bioresources and Bioprocessing (2022) 9:33 Page 8 of 10 Fig. 3 Molecular docking of pFPA with F-BbAmDH (a) and cFLF-AmDH (b). Substrate pFPA is shown as yellow sticks. Key residue K90 is shown as cyan sticks and the aromatic substrate pFPA were selected as repre- sentative substrates and calculated binding energy. The results are shown in Table 6. Lower energy requirements for binding to cFLF-AmDH whether it was aliphatic ketone or aromatic substrates. Reduction reactions of cFLF‑AmDH with different coenzyme concentrations Chimeric amine dehydrogenase cFLF-AmDH showed higher catalytic activity (k /K ) for coenzyme affin - cat m ity. Consequently, the reductive amination reaction of cFLF-AmDH was carried out at a coenzyme concentra- tion of 0.05  mM and 0.5  mM with p-FPA as substrate. The reaction curves are shown in Fig.  5; the maximum Fig. 4 Comparison and analysis of differences between cFLF-AmDH reaction conversion reached 75% catalyzed by cFLF- and F-BbAmDH based on MD simulation. The RMSF results of AmDH, whereas, it only reached 50% catalyzed by parent cFLF-AmDH (black) and F-BbAmDH (red) are shown in the line chart. They were calculated for all frames in the trajectory F-AmDH with 0.05  mM of NAD . When the coenzyme concentration increased to 0.5 mM, it took about 4 h for cFLF-AmDH to reach the 1 conversion of 100%, whereas, it took about 6  h for F-BbAmDH. The reaction results Table 6 The binding energies (ΔG ) of substrates and enzyme bind agreed with the previous dynamic parameter results of Substrates ΔG (kcal/mol) cFLF-AmDH for NADH. Thus, by increasing the cata - bind lytic efficiency of the enzyme for the coenzyme, the reac - F‑BbAmDH cFLF‑ AmDH tion proceeded more efficiently. pFPA − 5.9 − 8.2 2-Pentanone − 4.8 − 5.5 Conclusion In summary, to reduce the cost of industrial applications of biocatalysts and to improve the utilization efficiency fluctuation (RMSF) is useful for characterizing local of enzymes for coenzymes, we rationally designed and changes along the protein chain. According to the RMSF created a high NADH-affinity chimeric amine dehydro - value, the structure of cFLF-AmDH was more stable. The genase cFLF-AmDH using coenzyme-binding structural cFLF-AmDH model can be used as a promising template analysis. This led to a decrease in K of the enzyme for to produce chiral amines through a semi-rational design. NADH from 0.86 to 0.49  mM, with the reaction time Meanwhile, the aliphatic ketone substrate 2-pentanone shortened from 6 to 4  h. To gain deeper insight, MD Li  et al. Bioresources and Bioprocessing (2022) 9:33 Page 9 of 10 + + + Fig. 5 Eec ff t of NAD concentration on the reductive amination of pFPA. a 0.05 mM NAD ; b 0.5 mM NAD . Reaction conditions: pFPA (0.2 M), −1 NH Cl–NH H O (2 M), glucose (0.24 M) and purified cFLF-AmDH and F-BbAmDH. (1 gL ) in Tris–HCl buffer (0.1 M, pH 8.5), 30 °C, 200 rpm 4 3 2 Acknowledgements simulation analysis was used to explore the binding rela- Not applicable. tionship between the coenzymes and enzymes in sub- strate catalysis. The results showed that the chimeric Authors’ contributions XQM and TW contributed in “idea and overall outline of the work”. JLL curated amine dehydrogenase cFLF-AmDH we constructed had and performed all the experiments and written the manuscript. All authors a more stable structure and shortened distance between contributed to data analysis and proof-reading of the manuscript. All authors the key residue sites for substrate binding. These findings read and approved the final manuscript. provide a good basis for the industrial application of this Funding enzyme. The strategy employed in this study can also be This work was supported by the National Key Research and Development used to discover other enzymes with specific functions Program of China (Grant Numbers 2021YFC2100100), the National Natural Sci- ence Foundation of China (NSFC) (Grant Numbers 21336009 and 21176103), and to improve the efficiency of coenzyme utilization by and the National First-Class Discipline Program of Light Industry Technology oxidoreductases. and Engineering (Grant Number LITE2018-09). Availability of data and materials Abbreviations All data generated or analyzed during this study are included in this published F-AmDH: Phenylalanine amine dehydrogenase; cFLF-AmDH: Chimeric article and its supplementary information files. amine dehydrogenase; L-AmDH: Leucine amine dehydrogenase; pFPA: P-Fluorophenylacetone. Declarations Supplementary Information Ethics approval and consent to participate Not applicable. The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s40643- 022- 00528-0. Consent for publication All authors approved to publish to article. Additional file 1: Figure S1. Sequence Alignment. Amino acid sequence alignment of F-BbAmDH, L-BcAmDH, L-EsAmDH, and BsLeuDH. Align- Competing interests ment was performed using the MUSCLE server (https:// www. ebi. ac. uk/ The authors declare that they have no competing interests. Tools/ msa/ muscle/) and displayed using Esprit (http:// espri pt. ibcp. fr). Secondary structure elements are shown based on the BsLeuDH structure. Author details Protein structure is predicted by Robetta server (https:// robet ta. baker lab. Laboratory of Brewing Microbiology and Applied Enzymology, School org/). Figure S2. Structure comparison. Docking of proteins to ligands of Biotechnology, Jiangnan University, Wuxi 214122, China. K ey Laborator y was obtained with Auto Dock Tools (http:// autod ock. scrip ps. edu/ resou of Industrial Biotechnology, Ministry of Education, School of Biotechnology, rces/ adt). Protein structure maps were produced by the 3D visualization Jiangnan University, Wuxi 214122, China. Suqian Jiangnan University Institute software Pymol (https:// www. pymol. org). FBbAmDH is shown as green of Industrial Technology, Suqian 223800, China. cartoon, L-BcAmDH is shown as orange cartoon, LEsAmDH is shown as pink cartoon. Figure S3. SDS-PAGE analysis the cell-free extract of Received: 6 January 2022 Accepted: 17 March 2022 cFLFAmDH using LB medium (A), SDS-PAGE analysis the cell-free extract of cFLFAmDH using autoinduction medium (B). A: M, molecular weight marker, Lane 1 ~ 2, the cell-free extract of cFLFAmDH, Lane 3 ~ 4, broken centrifugal sediment of cFLFAmDH. B: M, molecular weight marker, Lane 1 ~ 3, the cell-free extract of cFLFAmDH, Lane 4 ~ 6, purified enzymes of cFLFAmDH. Li et al. Bioresources and Bioprocessing (2022) 9:33 Page 10 of 10 References Meng X, Yang L, Liu Y, Wang H, Shen Y, Wei D (2021) Identification and rational Abrahamson MJ, Vazquez-Figueroa E, Woodall NB, Moore JC, Bommarius AS engineering of a high substrate-tolerant leucine dehydrogenase effective (2012) Development of an amine dehydrogenase for synthesis of chiral for the synthesis of L-tert-leucine. ChemCatChem 13(14):3340–3349 amines. Angew Chem Int Ed Engl 51(16):3969–3972 Mu X, Wu T, Mao Y, Zhao Y, Xu Y, Nie Y (2021) Iterative alanine scanning Abrahamson MJ, Wong JW, Bommarius AS (2013) The evolution of an amine mutagenesis confers aromatic ketone specificity and activity of L-amine dehydrogenase biocatalyst for the asymmetric production of chiral dehydrogenases. ChemCatChem 13(24):5243–5253 amines. Adv Synth Catal 355(9):1780–1786 Robert X, Gouet P (2014) Deciphering key features in protein structures Bommarius BR, Schurmann M, Bommarius AS (2014) A novel chimeric amine with the new ENDscript server. Nucleic Acids Res 42( Web Server dehydrogenase shows altered substrate specificity compared to its par - issue):W320–W324 ent enzymes. Chem Commun (camb) 50(95):14953–14955 Ruffoni A, Julia F, Svejstrup TD, McMillan AJ, Douglas JJ, Leonori D (2019) Practi- Cai R-F, Liu L, Chen F-F, Li A, Xu J-H, Zheng G-W (2020) Reductive amina- cal and regioselective amination of arenes using alkyl amines. Nat Chem tion of biobased levulinic acid to unnatural chiral γ-amino acid 11(5):426–433 using an engineered amine dehydrogenase. ACS Sustain Chem Eng Sharkey MA, Engel PC (2009) Modular coenzyme specificity: a domain- 8(46):17054–17061 swopped chimera of glutamate dehydrogenase. Proteins 77(2):268–278 Chen F-F, Liu Y-Y, Zheng G-W, Xu J-H (2015) Asymmetric amination of second- Sharma M, Mangas-Sanchez J, Turner NJ, Grogan G (2017) NAD(P)H-depend- ary alcohols by using a redox-neutral two-enzyme cascade. Chem- ent dehydrogenases for the asymmetric reductive amination of ketones: CatChem 7(23):3838–3841 structure, mechanism, evolution and application. Adv Synth Catal Chen FF, Zheng GW, Liu L, Li H, Chen Q, Li FL, Li CX, Xu JH (2018) Reshaping 359(12):2011–2025 the active pocket of amine dehydrogenases for asymmetric synthesis of Son HF, Kim IK, Kim KJ (2015a) Structural insights into domain movement and bulky aliphatic amines. ACS Catal 8(3):2622–2628 cofactor specificity of glutamate dehydrogenase from Corynebacterium Ducrot L, Bennett M, Grogan G, Vergne-Vaxelaire C (2020) NAD(P)H-dependent glutamicum. Biochem Biophys Res Commun 459(3):387–392 enzymes for reductive amination: active site description and carbonyl- Son M, Park C, Kwon SG, Bang WY, Kim SW, Kim CW, Lee KW (2015b) Structural containing compound spectrum. Adv Synth Catal 363(2):328–351 importance of the C-terminal region in pig aldo-keto reductase family 1 Franklin RD, Mount CJ, Bommarius BR, Bommarius AS (2020) Separate sets of member C1 and their effects on enzymatic activity. BMC Struct Biol 15:1 mutations enhance activity and substrate scope of amine dehydroge- Tseliou V, Masman MF, Bohmer W, Knaus T, Mutti FG (2019) Mechanistic insight nase. ChemCatChem 12(9):2436–2439 into the catalytic promiscuity of amine dehydrogenases: asymmetric syn- Ganjoo A, Tripathi A, Chetti P (2020) Structural assessment and identification thesis of secondary and primary amines. ChemBioChem 20(6):800–812 of 11beta-hydroxysteroid dehydrogenase type 1 inhibitors. J Biomol Tseliou V, Knaus T, Vilim J, Masman MF, Mutti FG (2020) Kinetic resolution of Struct Dyn 38(16):4928–4937 racemic primary amines using Geobacillus stearothermophilus amine Genheden S, Ryde U (2015) The MM/PBSA and MM/GBSA methods to esti- dehydrogenase variant. ChemCatChem 12(8):2184–2188 mate ligand-binding affinities. Expert Opin Drug Discov 10(5):449–461 Wang JB, Reetz MT (2015) Biocatalysis: chiral cascades. Nat Chem Grogan G (2018) Synthesis of chiral amines using redox biocatalysis. Curr Opin 7(12):948–949 Chem Biol 43:15–22 Wang TD, Ma F, Ma X, Wang P (2015) Spatially programmed assembling of oxi- Huang H, Liu X, Zhou L, Chang M, Zhang X (2016) Direct asymmetric reductive doreductases with single-stranded DNA for cofactor-required reactions. amination for the synthesis of chiral beta-arylamines. Angew Chem Int Ed Appl Microbiol Biotechnol 99(8):3469–3477 Engl 55(17):5309–5312 Wu T, Mu X, Xue Y, Xu Y, Nie Y (2021) Structure-guided steric hindrance Hyndman RJ, Koehler AB (2006) Another look at measures of forecast accuracy. engineering of Bacillus badius phenylalanine dehydrogenase for efficient Int J Forecast 22(4):679–688 L-homophenylalanine synthesis. Biotechnol Biofuels 14(1):207 Itoh N, Yachi C, Kudome T (2000) Determining a novel NAD -dependent Zhou J, Wang Y, Chen J, Xu M, Yang T, Zheng J, Zhang X, Rao Z (2019) Rational amine dehydrogenase with a broad substrate range from Streptomyces engineering of Bacillus cereus leucine dehydrogenase towards alpha-keto Õirginiae IFO 12827: purification and characterization. J Mol Catal B acid reduction for improving unnatural amino acid production. Biotech- Enzym 10(1–3):281–290 nol J 14(3):e1800253 Jiang W, Fang B (2020) Synthesizing chiral drug intermediates by biocatalysis. Zhu L, Wu Z, Jin JM, Tang SY (2016) Directed evolution of leucine dehydroge- Appl Biochem Biotechnol 192(1):146–179 nase for improved efficiency of L-tert-leucine synthesis. Appl Microbiol Jiang W, Wang Y (2020) Improving catalytic efficiency and changing substrate Biotechnol 100(13):5805–5813 spectrum for asymmetric biocatalytic reductive amination. J Microbiol Zoi I, Antoniou D, Schwartz SD (2017) Incorporating fast protein dynamics into Biotechnol 30(1):146–154 enzyme design: a proposed mutant aromatic amine dehydrogenase. J Kataoka K, Tanizawa K (2003) Alteration of substrate specificity of leucine Phys Chem B 121(30):7290–7298 dehydrogenase by site-directed mutagenesis. J Mol Catal B Enzym 23(2–6):299–309 Publisher’s Note Kataoka K, Tanizawa K, Esaki N, Yoshimura T, Takada H (1994) Construction and Springer Nature remains neutral with regard to jurisdictional claims in pub- characterization of chimeric enzyme consisting of an amino-terminal lished maps and institutional affiliations. domain of phenylalanine dehydrogenase and a carboxy-terminal domain of leucine dehydrogenase. J Biochem 116(4):931–936 Le QA, Joo JC, Yoo YJ, Kim YH (2012) Development of thermostable Candida antarctica lipase B through novel in silico design of disulfide bridge. Biotechnol Bioeng 109(4):867–876 Li J, Pan J, Zhang J, Xu J-H (2014) Stereoselective synthesis of l-tert-leucine by a newly cloned leucine dehydrogenase from Exiguobacterium sibiricum. J Mol Catal B Enzym 105:11–17 Li FL, Zhou Q, Wei W, Gao J, Zhang YW (2019) Switching the substrate specific- ity from NADH to NADPH by a single mutation of NADH oxidase from Lactobacillus rhamnosus. Int J Biol Macromol 135:328–336 Madeira F, Park YM, Lee J, Buso N, Gur T, Madhusoodanan N, Basut- kar P, Tivey ARN, Potter SC, Finn RD, Lopez R (2019) The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res 47( W1):W636–W641 Mayol O, David S, Darii E, Debard A, Mariage A, Pellouin V, Petit J-L, Salanoubat M, de Berardinis V, Zaparucha A, Vergne-Vaxelaire C (2016) Asymmetric reductive amination by a wild-type amine dehydrogenase from the ther- mophilic bacteria Petrotoga mobilis. Catal Sci Technol 6(20):7421–7428 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioresources and Bioprocessing Springer Journals

High coenzyme affinity chimeric amine dehydrogenase based on domain engineering

Loading next page...
 
/lp/springer-journals/high-coenzyme-affinity-chimeric-amine-dehydrogenase-based-on-domain-blpmMIZUXY
Publisher
Springer Journals
Copyright
Copyright © The Author(s) 2022
eISSN
2197-4365
DOI
10.1186/s40643-022-00528-0
Publisher site
See Article on Publisher Site

Abstract

*Correspondence: xqmu@jiangnan.edu.cn Jialin Li and Xiaoqing Mu contributed equally Laboratory of Brewing Microbiology and Applied Enzymology, School of Biotechnology, Jiangnan University, Wuxi 214122, China Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Li et al. Bioresources and Bioprocessing (2022) 9:33 Page 2 of 10 140 to the terminus 366 by the L-AmDH from Bacil- Introduction lus stereothermophilus) via domain shuffling, which can Chiral amines are an important class of chiral building catalyze adamantyl methyl ketone to adamantyl ethyl- blocks that are widely used in fine chemicals, agriculture, amine unlike parent proteins and strongly improve ther- biologically active natural products, and pharmaceuti- mal activity. Ch1-AmDH (a chimeric enzyme obtained cal intermediates (Jiang and Fang 2020; Wang and Reetz through domain shuffling of first-generation variants) 2015). Compared with most chemical synthesis path- was used to understand the catalytic mechanism and ways that require harsh reaction conditions such as high the molecular discriminants that are crucial for the effi - temperature and high pressure, enzyme catalysis synthe- cient catalytic activity of AmDHs in 2019 (Tseliou et  al. sizes chiral chemicals through its high stereo selectivity, 2019). Crystal structure studies then on AmDHs sug- mild reaction conditions, and environmental friendli- gested that the monomer structure of AmDH consists of ness (Abrahamson et  al. 2012; Chen et  al. 2018; Sharma two independent substrate-binding and cofactor-binding et al. 2017). Among the enzymes that have been reported domains (Son et  al. 2015a). Subsequently, a chimeric for the synthesis of chiral amines, AmDH only requires enzyme that changes the specificity of the coenzyme has cheap ammonium ions as the amino donor, with water as been reported. By replacing the cofactor NA D bind- its only by-product, considered a greener synthetic route ing domain from Clostridium symbiotic to the cofactor (Chen et al. 2015; Huang et al. 2016). NADP binding domain of glutamate dehydrogenase At present, there are two main types of AmDH: natu- from Escherichia coli, the coenzyme dependency was ral AmDH and engineered AmDH modified from natural + + changed from NADP to NAD (Sharkey and Engel, amino acid dehydrogenase (Tseliou et  al. 2020). How- 2009). This chimeric enzyme showed NAD dependence ever, natural AmDH has few origins, a narrow substrate and high catalytic efficiency. This strategy suggests that spectrum, and low catalytic activity, greatly limiting their the combination or substitution of enzyme domains may application in the synthesis of chiral amines (Mayol et al. contribute to the overall property changes. 2016). Since its creation by Abrahamson et  al. (2012) in Recently, phenylalanine amine dehydrogenases that 2012, worldwide attention has focused on improving the catalyze difficult aromatic ketone substrates, important substrate spectrum and increasing the catalytic efficiency precursors of pharmaceutical intermediates, have been by rationally designing a directed evolution-engineered gradually reported (Abrahamson et  al. 2013; Ruffoni AmDH. Most of the research was aimed at modifying et  al. 2019; Zoi et  al. 2017). However, their lower coen- the site near the substrate-binding pocket to improve the zyme affinity is limited in industrial production (Kataoka properties of AmDH (Ducrot et  al. 2020; Grogan 2018; and Tanizawa 2003; Li et al. 2014; Zhu et al. 2016). Here, Itoh et al. 2000; Wu et al. 2021). As these are coenzyme- We report a chimeric enzyme, cFLF-AmDH, based on dependent enzymes, reaction efficiency can be improved homologous sequence alignment and structural analysis using a higher coenzyme concentration or increasing of the independent substrate and coenzyme of AmDH. the affinity between the enzyme and coenzyme (Frank - The coenzyme-binding domain of L-AmDH from Bacil - lin et  al. 2020; Itoh et  al. 2000; Zhou et  al. 2019). The lus cereus was used to replace the corresponding region latter improves the reaction efficiency and reduces the of F-AmDH from Bacillus badius, which has a lower dosage of expensive coenzymes to reduce production coenzyme affinity. The constructed cFLF-AmDH had costs. This strategy is gradually being applied (Cai et  al. high coenzyme affinity and catalytic efficiency, and fur - 2020; Jiang and Wang 2020; Li et  al. 2019; Wang et  al. ther broadened the substrate spectrum based on inherit- 2015). In 2021, by alignment with the coenzyme-binding ing the substrate specificity of F-BbAmDH. Subsequently, domain sequence of amino acid dehydrogenase which we used molecular dynamics (MD) simulations to clarify has higher coenzyme affinity, Meng et al. (2021) adjusted the factors that help to increase the coenzyme affinity the coenzyme-binding cavity increasing the NADH activ- and catalytic efficiency observed in kinetic analysis and ity of leucine dehydrogenase (LaLeuDH) from Labren- conversion experiments. This enables the rational design zia aggregata by introducing a double mutation in the of coenzyme-binding domains to screen good candidates coenzyme-binding region, thereby increasing reaction for improved catalytic efficiency and cofactor affinity. efficiency. With the continuous deepening of research, the modi- Materials and methods fication of AmDH is not limited to directed evolution. Strains, plasmids, and chemicals Domain shuffling provides an alternative method for F-BbAmDH from Bacillus badius、L-BcAmDH from obtaining enzymes with improved properties (Kataoka Bacillus cereus and L-EsAmDH from Exiguobacte- et  al. 1994). In 2014, Bommarius et  al. (2014) created a rium sibiricum were generated in the laboratory. Glu- novel chimeric amine dehydrogenase (residues 1–149 cose dehydrogenase from Bacillus amyloliquefaciens were contributed by F-AmDH from Bacillus badius and Li  et al. Bioresources and Bioprocessing (2022) 9:33 Page 3 of 10 (BaGluDH) was purchased from Sigma-Aldrich Corp. Kinetic parameter determination (Beijing, China). Escherichia coli BL21 (DE3) and plas- Kinetic parameters of parental F-BbAmDH and cFLF- mid pET-28a (+) were purchased from Novagen (Nan- AmDH were determined in N H Cl/NH OH buffer 4 4 jing, China) as the gene expression host and vector, (100 mM, pH 8.5) at 30 °C in different concentrations of respectively. p-Fluorophenyl acetone (p-FPA) was bought p-FPA (with concentration range from 0.05 to 50 mM) or from J&K Co. Ltd. (Shanghai, China). Isopropyl-β-D- NADH (with concentration range from 0.01 to 0.5 mM). + −1 −1 thiogalactoside (IPTG), kanamycin, NADH, and NAD The kinetic constant (K , mmol·L ; k, min ) and m cat −1 −1 −1 were from TCI (Shanghai, China). All other chemicals catalytic efficiency (k /K, L ·min ·mmol ) were cat m were analytical grade and commercially available. calculated using a nonlinear curve fitting of initial veloc - ity versus substrate concentration data to the Michaelis– Construction of cFLF‑AmDH expression vectors Menten equation by Origin software. All the values were The coenzyme-binding domains of two amine dehydro - averaged from three replicates with standard deviations. genases were predicted using the NCBI (https:// www. ncbi. nlm. nih. gov). Chimeric amine dehydrogenase Enzyme activity assay (cFLF-AmDH) was introduced using homologous recom- Enzyme activity was determined by monitoring the bination technology containing the coenzyme-binding change in absorbance at 340  nm at 30  °C using a Multi- domain of L-BcAmDH and the substrate-binding domain Skan GO UV-spectrometer (Thermo Fisher Scientific), of F-BbAmDH. The corresponding primers were syn - which corresponds to the change in the concentration thesized by Shenggong Bioengineering (Shanghai) Co., of NADH (Li et  al. 2014). For reductive amination, the Ltd. (Shanghai China). The PCR program was run for reaction mixture (200 μL) contained 20  mM substrate, 30 cycles under the following conditions: 30  s at 98  °C, 0.2  mM NADH, 2  M NH Cl/NH OH buffer (pH 9.0) 4 4 10  s at 98  °C, 30  s at 55  °C, 70  s at 72  °C, and 10  min at and a certain amount of purified enzyme. Enzyme activ - 72  °C, after which it was kept at 10  °C. The constructed ity unit (U) was defined as the amount of enzyme which plasmids were then isolated and sequenced prior to their catalyzes the production (or consumption) of 1  µmol of introduction into E. coli BL21 (DE3). The chimeric strain NADH per min under the above conditions. E. coli BL21/pET-28a-cFLFAmDH was obtained after confirmed by DNA sequencing. Enzymatic properties determination The enzyme activity of F-BbAmDH and cFLF-AmDH Expression and purification of enzymes was measured at various pH (7.0–11.0) in NH Cl/ Phenylalanine amine dehydrogenase (F-BbAmDH) NH OH buffer (30 °C). The pH stability was determined was cultured in Luria–Bertani (LB) medium contain- by enzymatic incubation in N H Cl/NH OH buffer at dif - 4 4 −1 ing kanamycin (50  mg·L ) at 37  °C and with shaking ferent pH (7.0–11.0) at 30 °C for 2 h. at 200  rpm for 2  h. Protein expression was induced by The enzyme activity was measured and calculated at the addition of IPTG at a final concentration of 0.2  mM various temperatures (30–70 °C) in 2 M NH Cl/NH OH 4 4 when the bacteria reached an OD value of 0.6–0.8, buffer (pH 9.0). Thermal stability of F-BbAmDH and and the cells were then cultured at 17  °C and 200  rpm cFLF-AmDH was characterized by half-life. The half-life for 12 h. cFLF-AmDH was cultured in an auto-induction (t ) was calculated by incubating in 2 M NH Cl/NH OH 1/2 4 4 medium at 37 °C with shaking at 200 rpm for 2 h. When buffer (pH 9.0) at 55  °C for different time. The t value 1/2 the OD value of culture was 0.6–0.8, the temperature at 55  °C was calculated using the following formula: −1 was adjusted to 17  °C to induce protein expression and t = ln2 k (k is the first-order rate constant, which is 1/2 cultured for 60  h. The culture was collected by centrifu - derived from the semi-log plot of incubation time and gation at 4  °C and 8000  rpm for 5  min and then stored residual activity) (Le et  al. 2012). Specific activity before at −  80  °C until further use. Proteins were expressed in incubation was normalized as 100%. E. coli BL21 (DE3) with His -tag at the C-terminus. Cells were lysed by ultrasonic cell crusher and the supernatant Biotransformation and analytical methods was collected by centrifugation at 12,000 × g for 30  min The reaction mixture (10  ml) consisted of 2  M NH Cl/ at 4 °C. Proteins were purified with an ÄKTA purifier sys - NH OH buffer (pH 8.5), 20  mM NAD , 2  mM P-FBA tem (GE Healthcare, Little Chalfont, UK) using Ni–NTA and 20 μg protein. The reaction was carried out at 37 °C affinity columns and Superdex 200 chromatography. The with shaking at 200  rpm for 24  h. Samples (1  mL) were enzyme purity was determined by SDS-PAGE. drawn at intervals and kept in boiling water for 10  min Li et al. Bioresources and Bioprocessing (2022) 9:33 Page 4 of 10 to terminate the reaction. The reaction liquid sample Results and discussion (1.0 ml) was extracted twice with 1.0 ml ethyl acetate, and Sequence and structure analysis of parent AmDHs 1.0 ml extract was taken for product determination. The F-BbAmDH from Bacillus badius (Abrahamson et  al. solutions were then passed through a 0.22-μm filter. 2013) is an engineered amine dehydrogenase that cata- The conversion of amine products were performed with lyzes aliphatic and aromatic ketone substrates. Compared GC-FID analysis on a 7890B GC (Agilent) using nitrogen with other reported engineered amine dehydrogenases, as the carrier gas. Analytic conditions: Grace Econo-Cap the natural affinity for coenzymes of F-BbAmDH is low, EC-WAX + column (30  m × 0.25  mm × 0.25  μm). Split increasing the cost of industrial production. Based on ratio 5:1, pressure 120.0  kPa. Column temperature pro- domain recombination technology, coenzyme domain −1 gram: starting at 90 °C, hold for 2 min, with 10 °C·min replacement is an effective means of improving parental to 180 °C, hold for 2 min. coenzyme affinity. Two high-affinity coenzyme domain donors (Table  1), Structure modeling, molecular docking, and MD L-BcAmDH from Bacillus cereus (Mu et  al. 2021) and simulation L-EsAmDH from Exiguobacterium sibiricum (Chen et  al. Amino acid sequence alignment was performed using the 2018), were used as donors to obtain a structure similar to MUSCLE server (https:// www. ebi. ac. uk/ Tools/ msa/ mus- that of the amino acid dehydrogenase superfamily (Li et  al. cle/) (Madeira et al. 2019) and displayed using the Esprit 2014). All  three  enzymes  were  derived  from the  introduc- server (https:// espri pt. ibcp. fr/ ESPri pt/ ESPri pt/) (Rob- tion to KS/NL double mutations that alter the substrate spec- ert and Gouet 2014). Protein structure was predicted ificity at the catalytically  active  center of  the corresponding by Robetta server (https:// robet ta. baker lab. org/). Dock- amino  acid  dehydrogenases. To investigate the structural ing of proteins to ligands was obtained with Auto Dock homology of F-BbAmDH, L-BcAmDH, and L-EsAmDH, Tools (http:// autod ock. scrip ps. edu/ resou rces/ adt). Pro- the amino acid sequences, and tertiary structures were tein structure maps were produced by the 3D visualiza- compared using sequence alignment and Robetta server tion software Pymol (https:// www. pymol. org). Molecular modeling. The whole length of F-BbAmDH had 46.84% and dynamics (MD) simulations using GROMACS version 49.48% amino acid sequence homology with L-BcAmDH 5.0.2 and AMBER force field (Ganjoo et al. 2020). System and L-EsAmDH, respectively (Additional file  1: Figure S1). was built in a three-centered water model in an orthog- Meanwhile, F-BbAmDH shared 47.84% and 48.78% amino onal box that extends 10  Å from the dissolved atoms in acid sequence homology with L-BcAmDH and L-EsAmDH all three dimensions in order to create buffers between in the coenzyme-binding region, respectively. The three them. Use the steepest descent algorithm for energy enzymes were highly similar in their tertiary structure (Addi- minimization, a total of 10,000 steps (Son et  al. 2015b). tional file  1: Figure S2). L-BcAmDH was chosen as a cofactor In addition, the kinetics of the protein–coenzyme com- domain donor for the higher coenzyme affinity of approxi - plex and the protein–ligand complex were simulated for mately 2.5-fold compared with L-EsAmDH (Table 1). 50 ns and the system was heated to 300 K at a pressure of 1.01 bar (Ganjoo et al. 2020). Molecular dynamics simu- lation trajectories for binding free energy calculations Coenzyme affinity of chimeric amine dehydrogenase were performed using the MM/GBSA method, defined The structural study suggested that F-BbAmDH is ΔG = G -G -G (Genheden and Ryde 2015). composed of a substrate-binding domain, coenzyme- bind PL P L binding domain, and terminal structure. The chimeric Table 1 Kinetic parameters of the parent for substrate NADH Table 2 Kinetic parameters of chimeric amine dehydrogenase and F-BbAmDH for substrate NADH −1 Enzyme Substrate K (mM) k (min ) k /K m cat cat m −1 −1 (mM  min ) −1 Enzyme Substrate K (mM) k (min ) k /K m cat cat m −1 −1 (mM  min ) F-BbAmDH NADH 0.16 ± 0.02 0.65 ± 0.01 4.03 L-BcAmDH NADH 0.021 ± 0.004 1.09 ± 0.1 51.40 F-BbAmDH NADH 0.16 ± 0.02 0.65 ± 0.01 4.03 cFLFAmDH NADH 0.045 ± 0.005 1.42 ± 0.22 31.56 L-EsAmDH NADH 0.052 ± 0.005 1.62 ± 0.22 31.15 Determination of coenzyme kinetic parameters using the same substrate Comparison of coenzyme kinetics of cFLF-AmDH and F-BbAmDH. Determination 2-pentanone. The values were generated by fitting the initial specific activity of coenzyme kinetic parameters using the same substrate 2-pentanone. data to the Michaelis–Menten equation using nonlinear regression with The values were generated by fitting the initial specific activity data to the GraphPad Prism software. Value is means ± standard deviations. All reactions Michaelis–Menten equation using nonlinear regression with GraphPad Prism involved in the kinetic constant calculations were analyzed using a 2 M NH Cl/ software. At the same substrate concentration, the kinetic parameters of NH OH buffer at optimum pH and temperature. All experiments were repeated the coenzyme are measured using NADH of different concentrations, that is, 3 times 0.05–0.5 mM Li  et al. Bioresources and Bioprocessing (2022) 9:33 Page 5 of 10 amine dehydrogenase cFLF-AmDH was built from the stabilities were inversely proportional. The RMSD value substrate-binding domain and terminal structure from of the cFLF-AmDH complex was between F-BbAmDH F-BbAmDH and the coenzyme-binding domain from and L-BcAmDH, which was aligned with the measured L-BcAmDH. It was soluble (Additional file  1: Figure catalytic properties. The cFLF-AmDH did not fully char - S3), expressed by self-induction culture, and the kinetic acterize the full activity of L-BcAmDH. However, com- parameters of the coenzyme were determined accord- pared with F-BbAmDH, there was a significant change. ing to the Michaelis–Menten equation using nonlinear regression with GraphPad Prism software. Compared Enzymatic properties of cFLF‑AmDH with the F-BbAmDH, the k /K values for NADH of According to the results of the coenzyme affinity analy - cat m cFLF-AmDH increased by 4.2-fold. The k values were sis and MD simulation, the chimeric enzyme had better cat 2.2-fold higher and the K values were twofold lower properties. Subsequently, we investigated the enzymatic (Table 2). These results indicated that cFLF-AmDH had properties of cFLF-AmDH and F-BbAmDH. The optimal a stronger affinity and catalytic efficiency for NADH. reaction temperatures of cFLF-AmDH and F-BbAmDH were 60  °C and 55  °C, respectively (Fig.  2a). However, MD simulation of chimeric amine dehydrogenase‑NADH thermal optima of cFL1-AmDH and cFL2-AmDH (Bom- complexes marius et  al. 2014) were both greater than 70  °C. The To understand the molecular mechanism of the cata- thermal stabilities researched were carried out at 55  °C lytic efficiency improvement of cFLF-AmDH, all-atom by calculating the half-life (t ). The half-life of cFLF- 1/2 MD simulations for F-BbAmDH, L-BcAmDH, and AmDH was 9.6  h which was 160% higher than that of cFLF-AmDH as well as their complexes with NADH at parent F-BbAmDH (6.0 h; Fig. 2b and 2e) and the half-life 300  K were performed to analyze the structural changes of cFL1-AmDH (Bommarius et al. 2014) also greater than that occur in proteins. The root-mean-square deviation 500 min at 55 °C. (RMSD) was used to measure the average deviation of the The optimal reaction pH value and stability of protein conformation from the original structure (Hynd- F-BbAmDH and cFLF-AmDH were similar (Fig.  2c and man and Koehler 2006). As shown in Fig.  1, the RMSD 2d). Although the optimal reaction pH value was 10.0, evolution of the complex of F-BbAmDH and NADH the residual activity after 2  h of incubation decreased as showed that the variation range was 2.5–3.5  Å  under the pH value increased. It retained more than 80% and Cα, 3.0–4.5 Å under the side chain, and 4.0–5.5 Å under only 20–25% activity at a neutral pH (7.0) and alkaline the heavy atom. The RMSD evolution of the L-Bc AmDH pH (10.0), respectively. and NADH complexes also showed that Cα, side chains, heavy atoms varied between 1.5–2.0  Å, 2.0–2.5  Å, and Substrate specificity of cFLF‑AmDH 2.5–3.5  Å, respectively. However, the trajectory of the Structurally, part of the amino acid residues in the coen- complex of cFLF-AmDH and NADH changed in the zyme-binding domain will participate in the formation of range of 1.5–2.5  Å under Cα, 2.5–3.5  Å under the side the substrate-binding pocket, so the replacement of the chain and 3.0–4.5 Å under heavy atoms. For composites coenzyme-binding domain may cause changes in sub- with similar structures, the RMSD values and structural strate specificity and activity. The activities of a series Fig. 1 Comparison and analysis of differences between F-BbAmDH, L-BcAmDH and cFLF-AmDH based on MD simulation. The above plot shows the RMSD evolution of F-BbAmDH (a), L-BcAmDH (b) and cFLF-AmDH (c) during the 50-ns simulation at 300 K and 1.01 bar pressure. The first frame is used as the reference. All protein frames are first aligned on the reference frame backbone, and then the RMSD of Cα (black), side chain (red), and heavy atoms (blue) were calculated Li et al. Bioresources and Bioprocessing (2022) 9:33 Page 6 of 10 Fig. 2 Comparison of enzymatic properties of cFLF-AmDH and F-BbAmDH. a Optimum reaction temperature. b Temperature stability. c Optimum reaction pH. d pH stability. e Half-life. The error bars showed the standard deviations of three replicates Li  et al. Bioresources and Bioprocessing (2022) 9:33 Page 7 of 10 −1 Table 3 Specific activity (mU mg ) of F-BbAmDH and cFLF- higher catalytic activity and broad substrate specificity AmDH toward aliphatic ketones than F-BbAmDH. The activity of cFLF-AmDH to 2-pen - −1 a tanone, p-fluorophenyl acetone (p-FPA), 4-methylpro - Substrates Specific activity (mU mg ) piophenone, and p-methoxypropiophenone increased by F‑BbAmDH cFLF‑ AmDH 500%, 250%, 200%, and 170%, respectively. cFLF-AmDH accepted new substrates such as 4-methyl-2-pentanone, 4-Methyl-2-butanone n.a. n.a 5-hydroxy-2-pentanone, acetophenone, and 3-methy- 4-Hydroxy-2-butanone n.a n.a lacetophenone. Our cFLF-AmDH had activity against 2-Pentanone 33.5 143.32 acetophenone and pFPA at 30 °C, however, cFL1-AmDH 4-Methyl-2-pentanone n.a 25.29 was hardly active at 30 °C (Bommarius et al. 2014). 5-Hydroxy-2-pentanone n.a 34.75 p-FPA was selected as the test substrate because pheny- Activity was measured in NH Cl/NH OH buffer (2 M, pH 9.0) containing 0.2 mM 4 4 lacetone, the simplest ketone analog of phenylpyruvate, NADH and 20 mM substrate at 30 °C n.a. = no measurable activity the natural substance of phenylalanine dehydrogenase, was not readily available as a controlled substance and its vitality increased the most in this study. To observe −1 Table 4 Specific activity (mU mg ) of F-BbAmDH and cFLF- the changes in coenzyme affinity and substrate affinity, AmDH toward aromatic ketones the kinetic parameters were determined with pFPA and −1 a Substrates Specific activity (mU mg ) NADH at various concentrations. The obtained kinetic data are summarized in Table  5. cFLF-AmDH yielded F‑BbAmDH cFLF‑ AmDH 1.7-fold lower K values and 1.2-fold increases in k val- m cat Acetophenone n.a. 21.96 ues. This led to a 2.1-fold increase in k /K values for cat m 2-Methylacetophenone n.a n.a NADH. Compared with F-BbAmDH, cFLF-AmDH gave 3-Methylacetophenone n.a 23.32 threefold higher k values and 2.6-fold higher K val- cat m 4-Methylacetophenone n.a n.a ues for pFPA reduction, resulting in a 1.2-fold increase in 4-Fluoroacetophenone n.a n.a the k /K values. These results indicated that the affin - cat m 3-Hydroxyacetophenone n.a n.a ity of cFLF-AmDH for coenzymes increased and that the 4-Methoxyacetophenone n.a n.a catalytic efficiency of the coenzyme and substrate pFPA 4-Fluorophenylacetone 719.8 1759.4 improved. For comparison with cFL1-AmDH and cFL2- 4-Methylpropiophenone 377.9 838.1 AmDH, we also measured coenzyme kinetic param- 4-Hydroxypropiophenone n.a n.a eters at 60  °C. The K and K values of cFLF-AmDH m cat −1 P-Methoxyphenylacetone 199.1 337.9 were 0.05  mM and 132.44  min , respectively, at 60  °C. 4-(4-Methoxyphenyl)-2-butanone n.a n.a Although the K value was not much different, the K m cat Activity was measured in NH Cl/NH OH buffer (2 M, pH 9.0) containing 0.2 mM value of cFLF-AmDH was 2.5-fold larger than that of 4 4 NADH and 20 mM substrate at 30 °C cFL1-AmDH (Bommarius et al. 2014). n.a. = no measurable activity In the docking analysis, the distance between p-FPA and the side chain of cFLF-AmDH-K90 was 2.0  Å. The K90 was the key residue for the interaction between of aliphatic ketone substrates and aromatic ketone sub- ammonia and the substrate (Fig.  3). MD simulation strates of cFLF-AmDH and F-BbAmDH were investi- studies the structural changes that occur when a sub- gated and compared. For both aliphatic (Table  3) and strate binds to a protein (Fig.  4). The root-mean-square aromatic (Table  4) ketone substrates, cFLF-AmDH had Table 5 Kinetic parameters of the F-BbAmDH and cFLF-AmDH Enzyme pFPA NADH −1 −1 K (mM) k (min ) k /K K (mM) k (min ) k /K m cat cat m m cat cat m −1 −1 −1 −1 (min  mM ) (min  mM ) F-BbAmDH 8.262 ± 1.13 56.74 ± 4.83 6.60 0.86 ± 0.05 47.66 ± 3.46 55.40 cFLF-AmDH 21.81 ± 2.67 173.4 ± 6.94 7.95 0.49 ± 0.02 56.91 ± 5.12 116.14 The values were generated by fitting the initial specific activity data to the Michaelis–Menten equation using nonlinear regression with GraphPad Prism software. Value is means ± standard deviations. All reactions involved in the kinetic constant calculations were analyzed using a 2 M NH Cl/NH OH buffer at optimum pH and 4 4 temperature. All experiments were repeated 3 times Li et al. Bioresources and Bioprocessing (2022) 9:33 Page 8 of 10 Fig. 3 Molecular docking of pFPA with F-BbAmDH (a) and cFLF-AmDH (b). Substrate pFPA is shown as yellow sticks. Key residue K90 is shown as cyan sticks and the aromatic substrate pFPA were selected as repre- sentative substrates and calculated binding energy. The results are shown in Table 6. Lower energy requirements for binding to cFLF-AmDH whether it was aliphatic ketone or aromatic substrates. Reduction reactions of cFLF‑AmDH with different coenzyme concentrations Chimeric amine dehydrogenase cFLF-AmDH showed higher catalytic activity (k /K ) for coenzyme affin - cat m ity. Consequently, the reductive amination reaction of cFLF-AmDH was carried out at a coenzyme concentra- tion of 0.05  mM and 0.5  mM with p-FPA as substrate. The reaction curves are shown in Fig.  5; the maximum Fig. 4 Comparison and analysis of differences between cFLF-AmDH reaction conversion reached 75% catalyzed by cFLF- and F-BbAmDH based on MD simulation. The RMSF results of AmDH, whereas, it only reached 50% catalyzed by parent cFLF-AmDH (black) and F-BbAmDH (red) are shown in the line chart. They were calculated for all frames in the trajectory F-AmDH with 0.05  mM of NAD . When the coenzyme concentration increased to 0.5 mM, it took about 4 h for cFLF-AmDH to reach the 1 conversion of 100%, whereas, it took about 6  h for F-BbAmDH. The reaction results Table 6 The binding energies (ΔG ) of substrates and enzyme bind agreed with the previous dynamic parameter results of Substrates ΔG (kcal/mol) cFLF-AmDH for NADH. Thus, by increasing the cata - bind lytic efficiency of the enzyme for the coenzyme, the reac - F‑BbAmDH cFLF‑ AmDH tion proceeded more efficiently. pFPA − 5.9 − 8.2 2-Pentanone − 4.8 − 5.5 Conclusion In summary, to reduce the cost of industrial applications of biocatalysts and to improve the utilization efficiency fluctuation (RMSF) is useful for characterizing local of enzymes for coenzymes, we rationally designed and changes along the protein chain. According to the RMSF created a high NADH-affinity chimeric amine dehydro - value, the structure of cFLF-AmDH was more stable. The genase cFLF-AmDH using coenzyme-binding structural cFLF-AmDH model can be used as a promising template analysis. This led to a decrease in K of the enzyme for to produce chiral amines through a semi-rational design. NADH from 0.86 to 0.49  mM, with the reaction time Meanwhile, the aliphatic ketone substrate 2-pentanone shortened from 6 to 4  h. To gain deeper insight, MD Li  et al. Bioresources and Bioprocessing (2022) 9:33 Page 9 of 10 + + + Fig. 5 Eec ff t of NAD concentration on the reductive amination of pFPA. a 0.05 mM NAD ; b 0.5 mM NAD . Reaction conditions: pFPA (0.2 M), −1 NH Cl–NH H O (2 M), glucose (0.24 M) and purified cFLF-AmDH and F-BbAmDH. (1 gL ) in Tris–HCl buffer (0.1 M, pH 8.5), 30 °C, 200 rpm 4 3 2 Acknowledgements simulation analysis was used to explore the binding rela- Not applicable. tionship between the coenzymes and enzymes in sub- strate catalysis. The results showed that the chimeric Authors’ contributions XQM and TW contributed in “idea and overall outline of the work”. JLL curated amine dehydrogenase cFLF-AmDH we constructed had and performed all the experiments and written the manuscript. All authors a more stable structure and shortened distance between contributed to data analysis and proof-reading of the manuscript. All authors the key residue sites for substrate binding. These findings read and approved the final manuscript. provide a good basis for the industrial application of this Funding enzyme. The strategy employed in this study can also be This work was supported by the National Key Research and Development used to discover other enzymes with specific functions Program of China (Grant Numbers 2021YFC2100100), the National Natural Sci- ence Foundation of China (NSFC) (Grant Numbers 21336009 and 21176103), and to improve the efficiency of coenzyme utilization by and the National First-Class Discipline Program of Light Industry Technology oxidoreductases. and Engineering (Grant Number LITE2018-09). Availability of data and materials Abbreviations All data generated or analyzed during this study are included in this published F-AmDH: Phenylalanine amine dehydrogenase; cFLF-AmDH: Chimeric article and its supplementary information files. amine dehydrogenase; L-AmDH: Leucine amine dehydrogenase; pFPA: P-Fluorophenylacetone. Declarations Supplementary Information Ethics approval and consent to participate Not applicable. The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s40643- 022- 00528-0. Consent for publication All authors approved to publish to article. Additional file 1: Figure S1. Sequence Alignment. Amino acid sequence alignment of F-BbAmDH, L-BcAmDH, L-EsAmDH, and BsLeuDH. Align- Competing interests ment was performed using the MUSCLE server (https:// www. ebi. ac. uk/ The authors declare that they have no competing interests. Tools/ msa/ muscle/) and displayed using Esprit (http:// espri pt. ibcp. fr). Secondary structure elements are shown based on the BsLeuDH structure. Author details Protein structure is predicted by Robetta server (https:// robet ta. baker lab. Laboratory of Brewing Microbiology and Applied Enzymology, School org/). Figure S2. Structure comparison. Docking of proteins to ligands of Biotechnology, Jiangnan University, Wuxi 214122, China. K ey Laborator y was obtained with Auto Dock Tools (http:// autod ock. scrip ps. edu/ resou of Industrial Biotechnology, Ministry of Education, School of Biotechnology, rces/ adt). Protein structure maps were produced by the 3D visualization Jiangnan University, Wuxi 214122, China. Suqian Jiangnan University Institute software Pymol (https:// www. pymol. org). FBbAmDH is shown as green of Industrial Technology, Suqian 223800, China. cartoon, L-BcAmDH is shown as orange cartoon, LEsAmDH is shown as pink cartoon. Figure S3. SDS-PAGE analysis the cell-free extract of Received: 6 January 2022 Accepted: 17 March 2022 cFLFAmDH using LB medium (A), SDS-PAGE analysis the cell-free extract of cFLFAmDH using autoinduction medium (B). A: M, molecular weight marker, Lane 1 ~ 2, the cell-free extract of cFLFAmDH, Lane 3 ~ 4, broken centrifugal sediment of cFLFAmDH. B: M, molecular weight marker, Lane 1 ~ 3, the cell-free extract of cFLFAmDH, Lane 4 ~ 6, purified enzymes of cFLFAmDH. Li et al. Bioresources and Bioprocessing (2022) 9:33 Page 10 of 10 References Meng X, Yang L, Liu Y, Wang H, Shen Y, Wei D (2021) Identification and rational Abrahamson MJ, Vazquez-Figueroa E, Woodall NB, Moore JC, Bommarius AS engineering of a high substrate-tolerant leucine dehydrogenase effective (2012) Development of an amine dehydrogenase for synthesis of chiral for the synthesis of L-tert-leucine. ChemCatChem 13(14):3340–3349 amines. Angew Chem Int Ed Engl 51(16):3969–3972 Mu X, Wu T, Mao Y, Zhao Y, Xu Y, Nie Y (2021) Iterative alanine scanning Abrahamson MJ, Wong JW, Bommarius AS (2013) The evolution of an amine mutagenesis confers aromatic ketone specificity and activity of L-amine dehydrogenase biocatalyst for the asymmetric production of chiral dehydrogenases. ChemCatChem 13(24):5243–5253 amines. Adv Synth Catal 355(9):1780–1786 Robert X, Gouet P (2014) Deciphering key features in protein structures Bommarius BR, Schurmann M, Bommarius AS (2014) A novel chimeric amine with the new ENDscript server. Nucleic Acids Res 42( Web Server dehydrogenase shows altered substrate specificity compared to its par - issue):W320–W324 ent enzymes. Chem Commun (camb) 50(95):14953–14955 Ruffoni A, Julia F, Svejstrup TD, McMillan AJ, Douglas JJ, Leonori D (2019) Practi- Cai R-F, Liu L, Chen F-F, Li A, Xu J-H, Zheng G-W (2020) Reductive amina- cal and regioselective amination of arenes using alkyl amines. Nat Chem tion of biobased levulinic acid to unnatural chiral γ-amino acid 11(5):426–433 using an engineered amine dehydrogenase. ACS Sustain Chem Eng Sharkey MA, Engel PC (2009) Modular coenzyme specificity: a domain- 8(46):17054–17061 swopped chimera of glutamate dehydrogenase. Proteins 77(2):268–278 Chen F-F, Liu Y-Y, Zheng G-W, Xu J-H (2015) Asymmetric amination of second- Sharma M, Mangas-Sanchez J, Turner NJ, Grogan G (2017) NAD(P)H-depend- ary alcohols by using a redox-neutral two-enzyme cascade. Chem- ent dehydrogenases for the asymmetric reductive amination of ketones: CatChem 7(23):3838–3841 structure, mechanism, evolution and application. Adv Synth Catal Chen FF, Zheng GW, Liu L, Li H, Chen Q, Li FL, Li CX, Xu JH (2018) Reshaping 359(12):2011–2025 the active pocket of amine dehydrogenases for asymmetric synthesis of Son HF, Kim IK, Kim KJ (2015a) Structural insights into domain movement and bulky aliphatic amines. ACS Catal 8(3):2622–2628 cofactor specificity of glutamate dehydrogenase from Corynebacterium Ducrot L, Bennett M, Grogan G, Vergne-Vaxelaire C (2020) NAD(P)H-dependent glutamicum. Biochem Biophys Res Commun 459(3):387–392 enzymes for reductive amination: active site description and carbonyl- Son M, Park C, Kwon SG, Bang WY, Kim SW, Kim CW, Lee KW (2015b) Structural containing compound spectrum. Adv Synth Catal 363(2):328–351 importance of the C-terminal region in pig aldo-keto reductase family 1 Franklin RD, Mount CJ, Bommarius BR, Bommarius AS (2020) Separate sets of member C1 and their effects on enzymatic activity. BMC Struct Biol 15:1 mutations enhance activity and substrate scope of amine dehydroge- Tseliou V, Masman MF, Bohmer W, Knaus T, Mutti FG (2019) Mechanistic insight nase. ChemCatChem 12(9):2436–2439 into the catalytic promiscuity of amine dehydrogenases: asymmetric syn- Ganjoo A, Tripathi A, Chetti P (2020) Structural assessment and identification thesis of secondary and primary amines. ChemBioChem 20(6):800–812 of 11beta-hydroxysteroid dehydrogenase type 1 inhibitors. J Biomol Tseliou V, Knaus T, Vilim J, Masman MF, Mutti FG (2020) Kinetic resolution of Struct Dyn 38(16):4928–4937 racemic primary amines using Geobacillus stearothermophilus amine Genheden S, Ryde U (2015) The MM/PBSA and MM/GBSA methods to esti- dehydrogenase variant. ChemCatChem 12(8):2184–2188 mate ligand-binding affinities. Expert Opin Drug Discov 10(5):449–461 Wang JB, Reetz MT (2015) Biocatalysis: chiral cascades. Nat Chem Grogan G (2018) Synthesis of chiral amines using redox biocatalysis. Curr Opin 7(12):948–949 Chem Biol 43:15–22 Wang TD, Ma F, Ma X, Wang P (2015) Spatially programmed assembling of oxi- Huang H, Liu X, Zhou L, Chang M, Zhang X (2016) Direct asymmetric reductive doreductases with single-stranded DNA for cofactor-required reactions. amination for the synthesis of chiral beta-arylamines. Angew Chem Int Ed Appl Microbiol Biotechnol 99(8):3469–3477 Engl 55(17):5309–5312 Wu T, Mu X, Xue Y, Xu Y, Nie Y (2021) Structure-guided steric hindrance Hyndman RJ, Koehler AB (2006) Another look at measures of forecast accuracy. engineering of Bacillus badius phenylalanine dehydrogenase for efficient Int J Forecast 22(4):679–688 L-homophenylalanine synthesis. Biotechnol Biofuels 14(1):207 Itoh N, Yachi C, Kudome T (2000) Determining a novel NAD -dependent Zhou J, Wang Y, Chen J, Xu M, Yang T, Zheng J, Zhang X, Rao Z (2019) Rational amine dehydrogenase with a broad substrate range from Streptomyces engineering of Bacillus cereus leucine dehydrogenase towards alpha-keto Õirginiae IFO 12827: purification and characterization. J Mol Catal B acid reduction for improving unnatural amino acid production. Biotech- Enzym 10(1–3):281–290 nol J 14(3):e1800253 Jiang W, Fang B (2020) Synthesizing chiral drug intermediates by biocatalysis. Zhu L, Wu Z, Jin JM, Tang SY (2016) Directed evolution of leucine dehydroge- Appl Biochem Biotechnol 192(1):146–179 nase for improved efficiency of L-tert-leucine synthesis. Appl Microbiol Jiang W, Wang Y (2020) Improving catalytic efficiency and changing substrate Biotechnol 100(13):5805–5813 spectrum for asymmetric biocatalytic reductive amination. J Microbiol Zoi I, Antoniou D, Schwartz SD (2017) Incorporating fast protein dynamics into Biotechnol 30(1):146–154 enzyme design: a proposed mutant aromatic amine dehydrogenase. J Kataoka K, Tanizawa K (2003) Alteration of substrate specificity of leucine Phys Chem B 121(30):7290–7298 dehydrogenase by site-directed mutagenesis. J Mol Catal B Enzym 23(2–6):299–309 Publisher’s Note Kataoka K, Tanizawa K, Esaki N, Yoshimura T, Takada H (1994) Construction and Springer Nature remains neutral with regard to jurisdictional claims in pub- characterization of chimeric enzyme consisting of an amino-terminal lished maps and institutional affiliations. domain of phenylalanine dehydrogenase and a carboxy-terminal domain of leucine dehydrogenase. J Biochem 116(4):931–936 Le QA, Joo JC, Yoo YJ, Kim YH (2012) Development of thermostable Candida antarctica lipase B through novel in silico design of disulfide bridge. Biotechnol Bioeng 109(4):867–876 Li J, Pan J, Zhang J, Xu J-H (2014) Stereoselective synthesis of l-tert-leucine by a newly cloned leucine dehydrogenase from Exiguobacterium sibiricum. J Mol Catal B Enzym 105:11–17 Li FL, Zhou Q, Wei W, Gao J, Zhang YW (2019) Switching the substrate specific- ity from NADH to NADPH by a single mutation of NADH oxidase from Lactobacillus rhamnosus. Int J Biol Macromol 135:328–336 Madeira F, Park YM, Lee J, Buso N, Gur T, Madhusoodanan N, Basut- kar P, Tivey ARN, Potter SC, Finn RD, Lopez R (2019) The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res 47( W1):W636–W641 Mayol O, David S, Darii E, Debard A, Mariage A, Pellouin V, Petit J-L, Salanoubat M, de Berardinis V, Zaparucha A, Vergne-Vaxelaire C (2016) Asymmetric reductive amination by a wild-type amine dehydrogenase from the ther- mophilic bacteria Petrotoga mobilis. Catal Sci Technol 6(20):7421–7428

Journal

Bioresources and BioprocessingSpringer Journals

Published: Mar 27, 2022

Keywords: Amine dehydrogenase; Coenzyme affinity; Coenzyme binding domain; Catalytic efficiency

References