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Structure-guided protein engineering of ammonia lyase for efficient synthesis of sterically bulky unnatural amino acids

Structure-guided protein engineering of ammonia lyase for efficient synthesis of sterically bulky... Enzymatic asymmetric amination addition is seen as a promising approach for synthesizing amine derivatives, espe‑ cially unnatural amino acids, which are valuable precursors to fine chemicals and drugs. Despite the broad substrate spectrum of methylaspartate lyase (MAL), some bulky substrates, such as caffeic acid, cannot be effectively accepted. Herein, we report a group of variants structurally derived from Escherichia coli O157:H7 MAL (EcMAL). A combined mutagenesis strategy was used to simultaneously redesign the key residues of the entrance tunnel and binding pocket to explore the possibility of accepting bulky substrates with potential application to chiral drug synthesis. Libraries of residues capable of lining the active center of EcMAL were then constructed and screened by an effec‑ tive activity solid‑phase color screening method using tyrosinase as a cascade catalyst system. Activity assays and molecular dynamics studies of the resultant variants showed that the substrate specificity of Ec MAL was modified by adjusting the polarity of the binding pocket and the degree of flexibility of the entrance tunnel. Compared to M3, the optimal variant M8 was obtained with a 15‑fold increase in catalytic activity. This structure ‑based protein engineering of EcMAL can be used to open new application directions or to develop practical multi‑ enzymatic processes for the production of various useful compounds. Keywords: Biocatalysis, Methyl aspartate lyase, Substrate specificity, Protein engineering, Solid‑phase color screening Introduction of unnatural amino acids, having prominent advantages The hydroamination of olefins and carbon–nitrogen of economy and sustainability (Zhang et  al. 2020a). Six bond-forming reactions of unsaturated carboxylic acids classes of enzymes have been applied for this purpose, offer a vast range of applications in the synthesis of fine as exemplified by ω-transaminase (Mathew et  al. 2017; chemicals (Raj et  al. 2012). However, under classical Bea et  al. 2011), nitrilase (Yu et  al. 2019), amino acid conditions, the catalysts used are potentially hazard- dehydrogenase (Zhang et  al. 2015), ammonia lyase (AL) ous to the environment. With increasing emphasis on (Zhang et  al. 2020b), lipase (Zeng et  al. 2018), and tau- the concept of green, clean, and sustainable chemistry, tomerase (Liu et al. 2020). However, based on the princi- alternative strategies, such as biological methods, have ples of atomic-economy and cost-effectiveness, the most been proposed for implementing such reactions (Wu straightforward approach is the use of AL as the catalyst et  al. 2020). Enzymatic transformation of unsaturated to drive the hydroamination of unsaturated carboxylates carboxylic acids is an ideal approach for the production (Fibriansah et  al. 2011). This mild biocatalytic process would offer a welcome alternative for the synthesis of unnatural amino acids and their derivatives. ALs, belonging to the enolase family, specifically *Correspondence: wylou@scut.edu.cn Laboratory of Applied Biocatalysis, School of Food Science use unsaturated carboxylates as substrates for the and Engineering, South China University of Technology, No. 381 Wushan hydroamination reaction to generate unnatural amino Road, Guangzhou 510640, Guangdong, China © The Author(s) 2021. 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/. Ni et al. Bioresour. Bioprocess. (2021) 8:103 Page 2 of 9 acids and their derivatives (Turner 2011). Among Materials and methods ALs, MAL has attracted much attention because of its Chemicals broad substrate specificity and its acceptance of a vari- PrimerSTAR max DNA polymerase was obtained from ety of amines (Leese et  al. 2013). Recently, MAL and TaKaRa Biotechnology Co. (Dalian, China). Escherichia its variants have been reported to catalyze C–N bond coli strains BL21 (DE3) and DH5α were bought from formation in the synthesis of artificial dipeptide sweet- TransGen Biotech Co., Ltd. (Beijing, China). All other eners, showing that they possess broad amine scope in commercial chemicals were of analytical grade or above, accepting unnatural substrates (Zhang et  al. 2020b). and were purchased from Aladdin (Shanghai, China), However, despite these notable advances, unnatural Macklin (Shanghai, China), TCI (Tokyo, Japan) or Sigma- amino acids based on large-frame organic acids remain Aldrich (St. Louis, USA). largely unstudied. They can be envisaged as playing an important role in the synthesis of many chemicals, Homology modeling and molecular docking such as 3,4-dihydroxycinnamic acid, a precursor in the The EcMAL homology model was created using SWISS- production of levodopa (l -dopa) (Fordjour et al. 2019). MODEL (https:// swiss model. expasy. org/), taking MAL The potential of enzymes to be modified by directed from Clostridium tetanomorphum (PDB ID:1KKR) as a evolution has attracted much attention in recent years template (Lambrughi et  al. 2019). The modeled protein (Kramer et  al. 2019). Structure-guided protein engi- structure was evaluated through a Ramachandran plot, neering has replaced the natural mutation of enzymes and the percentage of residues in the allowed region was as one of the main tools of evolution (Cheng et  al. 98.6%. Molecular docking was performed with Yasara 2018; Renata et  al. 2015). For example, Wu and co- using the default program parameters. The center coor - workers used structure-based computational enzyme dinates of the box were determined by visual molecu- design to successfully convert aspartase into a series of lar dynamics, and the box size was set as 20  Å in each complementary hydroamination biocatalysts showing dimension. The docking results were selected according excellent regio- and enantioselectivity (Li et  al. 2018). to binding affinities and molecular conformations (Cheng However, the direction of directed evolution is uncon- et  al. 2018). PyMOL was used to display and analyze the trollable, so the correct selection of the mutagenesis modeled protein (Yuan et al. 2016). Amino acids with low strategies and the amount of screening verification conservation score at the entrance of the active pocket work are key in determining the ultimate degree of were chosen as hotspots (Yu et  al. 2019). CAVER 3.0 success (Zhou et al. 2016). Screening is the bottleneck (http:// www. caver. cz) was used to identify the tunnels of directed evolution, and two strategies are presently present in EcMAL (Zhang et al. 2019). in use. In the first, the focus is on developing efficient mutagenesis strategies to generate high-quality librar- PCR‑based methods for site mutagenesis and library ies to minimize the detection workload (Zhou et  al. construction of EcMAL 2016; Hu et al. 2020; Li et al. 2019). In the second, the Site-directed mutagenesis and combinatorial active site aim is to establish high-throughput screening meth- saturation testing were conducted by the overlap PCR ods, allowing rapid discrimination based on colorimet- and megaprimer approach with PrimerSTAR max DNA ric or optical changes (Li et al. 2019, 2020). polymerase. The reaction system had a total volume of 50 Our previous studies have already led to the mining μL, comprising 25 μL of PrimerSTAR max polymerase, of an MAL from E. coli O157:H7, which was found to 0.5 μL (50–100 ng) of template DNA, and 200 μm prim- catalyze the hydroamination of short-chain unsatu- ers mix (2 μL each), made up to the specified volume with rated acids such as fumaric acid or itaconic acid to the water. PCR amplification protocol for short fragments: corresponding amino acids with good selectivity. How- 98 °C for 2 min; (98 °C for 30 s, 55 °C for 30 s, 72 °C for ever, a dramatic decrease in reactivity was observed 1  min) × 30 cycles; 72  °C for 5  min. PCR products with when the substrate side chains increased. Therefore, the above short fragments were then used as primers using structural information as a guide to expand for mega-PCR: 98 °C for 2 min; (98 °C for 30 s, 60 °C for the substrate scope of engineering MALs is the main 30  s, 72  °C for 7  min) × 30 cycles; 72  °C for 10  min. The objective. In this study, we aimed to probe new resi- PCR products were resolved by agarose gel electropho- dues located near the active center and entrance tun- resis and purified using a Sangon Biotech purification nel, which may affect the acceptance of bulky aromatic kit. Digestion reaction program: 2 μL of NEB CutSmart unsaturated acids. We then further adjusted them to buffer and 1 μL of Dpn I were added for every 20 μL of expand the substrate spectrum of MAL. identified PCR product, and the mixture was incubated for 4  h at 37  °C. After thermal inactivation at 80  °C for N i et al. Bioresour. Bioprocess. (2021) 8:103 Page 3 of 9 20  min, plasmids containing the mutated gene were washed twice with 0.9% NaCl solution. The cells were directly transformed into E. coli BL21 (DE3) and then resuspended in buffer A (200  mM Tris–HCl buffer con - plated on a Luria–Bertani (LB) agar plate with 100 μg/mL taining 20  mM imidazole, 500  mM NaCl, pH 8.5) and of ampicillin. disrupted by ultrasonication. The cell lysates were cen - Multi-site simultaneous mutagenesis is based on spe- trifuged at 10,000g for 10  min at 4  °C to remove insolu- cific amino acid selection and primer design, which ble debris. The protein supernatant samples were loaded determines the size and quality of the constructed library. onto a HisTrap Ni–NTA FF column (GE Healthcare, Here, the 19 residues involved in EcMAL were divided USA) and eluted with buffer B (in which the imidazole into five groups (A–E). The short fragments between concentration was increased to 250  mM). The purified multiple mutation sites, as primers for the next round proteins were then further desalted by passage through a of amplification reactions, were then amplified by PCR desalination column (GE Healthcare, USA). The Bradford (Wang et  al. 2016). The purified short fragments were method was used to determine the protein concentration. used as primers to amplify the whole plasmid PET32a- EcMAL, leading to the final range of plasmids for library Specific activity and enantioselectivity assays generation. After digestion by Dpn I, the PCR products of the wild‑type and mutant enzymes were transformed into E. coli BL21 (DE3) cells to cre- The kinetic parameters of EcMAL were first measured ate a library for screening. Applying standard transfor- with varying concentrations of 1a (0.25–10 mM) and 1b mation procedures, the transformants were spread on (0.2–10  mM) as substrates by detecting the initial reac- HyBond-N membranes, which were then placed on the tion rate of the protein and curve-fitting according to the surface of LB/ampicillin (Amp, 100  μg/mL) agar plates. Michaelis–Menten equation. The specific activities and After incubation overnight at 37  °C, the membranes stereoselectivities of the EcMAL mutants toward 1a, 1b, were transferred to new LB/amp plates with 0.1  mM and 1c were then measured using purified enzymes. The isopropyl-β-d-thiogalactopyranoside (IPTG) and incu - reactions were carried out as follows: in a reaction sys- bated for 12 h at 30 °C. Each membrane was then trans- tem with a total volume of 2  mL, substrate 1a (10  mM), ferred to a clean plate and stored at – 20 °C prior to use. 1b (10 mM), or 1c (5 mM) in dimethyl sulfoxide (0.5 mL) and the purified enzyme were mixed with Tris–HCl Screening procedures (500  mM, pH 8.5). The mixture was shaken at 180  rpm Our solid-phase screening procedure is based on the and 30  °C, during which samples were withdrawn at analysis method devised by Turner’s group (Aleku et  al. regular intervals and extracted with an equal volume of 2017). Membranes containing the clones were repeat- ethyl acetate. The progress of the reaction was monitored edly frozen in liquid N and thawed four times to lyse the by HPLC analysis and all experiments were conducted in cells. The membranes were then placed on top of a filter triplicate. paper that was previously loaded with final concentra - 2+ tions of 10  mM screening substrate, 20  mM Mg , and Production of LOPA 500 mM NH Cl in 500  mM Tris–HCl buffer (pH 8.5). In a mixed system with a reaction volume of 20  mL, After incubation for 6 h at 30 °C, color-changed colonies the final concentrations of the components were as fol - were picked and inoculated into LB/amp for subsequent lows: 10 mM caffeic acid (with 2% (v/v) DMSO), 20 mM activity detection and mutant sequencing comparison. MgCl , 500  mM NH Cl, 200  mg/L purified enzyme, and 2 4 The number of mutants screened is calculated according 200  mM Tris–HCl buffer (pH 8.5). The reaction flask to the 95% mutation coverage determined by mutation was shaken at 180 rpm and 30 °C. Samples were regularly toolbox developed by Reezt’s team (Reetz et al. 2005). withdrawn and analyzed by HPLC. Protein expression and purification Results and discussion Wild-type and positive variants of EcMAL were inocu- Selection and grouping of key amino acids of Ecmal lated in 20  mL of LB medium containing 100  μg/mL of Based on our previous studies, we found that EcMAL ampicillin and incubated at 37 °C and 180 rpm. The over - could accept a range of short-chain unsaturated carbox- night cultures were transferred into 500  mL of medium ylic acids, including fumaric acid, mesaconic acid, and at a 1% inoculum level. When the absorbance of the itaconic acid, for the asymmetric synthesis of unnatural medium at 600 nm (OD600) reached 0.6–0.8, a final con - amino acids (Ni et  al. 2020). However, EcMAL showed centration of 0.1  mM IPTG was added, and the cultiva- low activity in the catalysis of bulky unsaturated car- tion temperature was adjusted to 16 °C to induce protein boxylic acids. This difference prompted us to investigate overexpression. After 20 h, each culture was harvested by the catalytic mechanism of EcMAL from a structural centrifuging at 8000g for 5  min, and the precipitate was perspective, with a view to further improve the catalytic Ni et al. Bioresour. Bioprocess. (2021) 8:103 Page 4 of 9 activity and stereoselectivity toward bulky substrates. We of degenerate NNK codons (N = A, G, C, T; K = G) for chose caffeic acid (1a) as the model compound for assess - saturation mutation would fully cover all possible amino ing the activity of EcMAL mutants, the basic reaction of acids, it is also accompanied by a dramatic increase in the which is shown in Additional file  1: Scheme S1. Four aro- number of screening (Wittmann et al. 2020). The degen - matic unsaturated carboxylic acids (cinnamic acid (1b), erate NDT codons (D = A, G, T) encoding the 12 differ - p-hydroxycinnamic acid (1c), methylcinnamic acid (1d), ent types of amino acids (C/F/H/D/N/S/I/G/V/L/Y/R) and acrylic acid (1e)) with different substituents were better meet the requirements of a “smart” library (Reetz also selected. These bulky compounds were selected as et  al. 2006). Thus, NDT was considered as a simplified substrates because their amination products are impor- codon for library construction. Because the combina- tant components or building blocks of therapeutic drugs, tion of simultaneous mutations increases exponentially, such as the Parkinson’s curative l-dopa. Considering that it is still necessary to screen at least 10 levels to achieve polyphenols can undergo a rapid color-forming reac- the theoretical 95% coverage (Wang et al. 2017). On this tion under the action of tyrosinase, we selected 1a as the basis, we developed a solid-phase screening method that screening substrate to assess the efficacies of the enzyme could rapidly screen 10 level transformants through variants. Based on the homologous three-dimensional color changes on a single plate. The single colonies that model of EcMAL, Yasara and CAVER 3.0 were used for met the requirements were subsequently selected for fur- molecular docking and substrate tunnel identification, ther activity testing (Tang et al. 2018). respectively (Liu et al. 2019; Heath et al. 2014). The sub - strate-binding pocket and substrate tunnel have been Screening and identification of active Ec mal variants demonstrated to have varying degrees of impact on the First of all, we designed mutation primers according to properties of the enzyme, thereby enhancing the differen - different mutation groups, and constructed the mutant tiation of directed evolution (De Raffele et  al. 2020). We libraries after amplification (Additional file  1: Figure simultaneously considered two groups of residues, those S1). Using the solid-phase color screening procedure lining the binding pocket (73, 170, 172, 194, 329, 384) and described in the experimental part, we performed high- those surrounding the substrate tunnel (196, 198, 199, throughput screening of five mutant libraries (Additional 240, 277, 307, 308, 356, 365, 389). Three residues (331, file  1: Figure S2). In the first round of screening, several 360, 361) participating in both regions were also taken mutants with activity to substrate 1a were screened from into account (Fig. 1). libraries A to E, and the location and type of mutations According to the type and spatial location of the 19 were further determined by sequencing. The screening selected residues, we attempted to divide them into five results are listed in Table  2 (M1–M7). Clearly, although groups, as shown in Fig. 2 and Table 1. Although the use the double mutations (M1–M3) showed new trace Fig. 1 EcMAL residues chosen for saturation mutagenesis marked in the homology model built by the crystal structure of MAL (PDB: 1KKR). A Active site mutation sites (yellow) selected on the basis of induced fit docking of amine 1 (green). B Residues surrounding the substrate access tunnel likewise chosen for mutagenesis (blue). The above residues were obtained through the molecular docking of Yasara and the channel recognition function of CAVER 3.0, respectively N i et al. Bioresour. Bioprocess. (2021) 8:103 Page 5 of 9 Fig. 2 Spatial structure information of substrate–enzyme docking. A The position relationship between EcMAL channels and the active sites. The target residues of libraries A–E are displayed in purple sticks. The substrate tunnel is shown in yellow and blue. B Poses of caffeic acid docked in the modeled structure of EcMAL. The substrate caffeic acid is shown in azure and the selected residues envelop the substrate in a surface view. The visualization of EcMAL channel, substrate and target amino acid residues was achieved by Pymol and Yasara Table 2 Properties of the EcMAL variants Table 1 Grouping of the 19 chosen EcMAL residues into five randomization sites (A, B, C, D and E) and the NDT codes used in Enzyme and mutants Mutation sites Specific % ee saturation mutagenesis activity (U/ g ) protein Randomization site Code c c WT EcMAL None nd nd M1 Q329S/K331R 5.4 ± 0.5 16 (S) A: 170, 172, 198, 199 NDT M2 Q73F/Y240C 4.4 ± 0.4 4.4 (S) B: 194, 196, 384, 389 NDT M3 F170L/E172F 2.5 ± 0.5 39 (S) M4 Q329S/K331G/C361C 6.2 ± 0.8 86(S) C: 307, 308, 356, 360 NDT M5 Q73V/Y240C/D277C 7.3 ± 0.1 28(S) M6 F170S/E172R/D307L 9.0 ± 0.3 79 (S) D: 329, 331, 361, 365 NDT M7 Q329F/K331R/Y356S/ 26.2 ± 0.2 99 (S) C361L E: 73, 240, 277 NDT M8 E172R/Y240S/D307L/ 38.6 ± 0.5 99 (S) Q329F/Y356S/C361L The red numbers denote the “active site” positions, the green ones as the “tunnel” positions, and blue ones as the “shared” positions Specific activity was determined at pH 8.0 and 30 °C using purified enzyme and omeprazole sulfide The ee values were determined by HPLC activity toward caffeic acid compared with the wild type, Not detected its activity and stereoselectivity were far from the expec- The bold mutations indicate newly involved mutation(s) in each round tations (Li et  al. 2019). However, the coincidence that the effective mutants were concentrated in libraries B, D, and E caught our attention. The triple mutants (M4– and confirming the specific activities, we identified the M6) screened by these libraries further indicated that the best variants (M8) toward the model substrates, which selected hot sites in libraries B, D, and E had a great influ - are shown in Table  2. Compared with the catalytic per- ence on the enzyme to accept bulky substrates. In par- formance identified by other mutants, the M8 presented ticular, a quadruple mutation M7 (Q329F/K331R/Y356S/ about 15-fold improvement and the selectivity was also C361L) suddenly exhibited a mutation transition effect, greatly improved. Considering that M8 was created by with remarkable improvements in catalytic activity and partial iterative mutation of M7, and the catalytic activity selectivity. Motivated by these results, M7 was chosen as was improved while the stereoselectivity was maintained, the parental enzyme for further iterative combinations. the results indicate a prominent synergistic influence Subsequently, a second round of screening experiments among these six residues. were implemented for libraries B and E using M7 as the To fully investigate the catalytic performance of the template. After screening approximately 5000 clones selected mutants, several substrates with varying sizes Ni et al. Bioresour. Bioprocess. (2021) 8:103 Page 6 of 9 and structures, including 1a, 1b, 1c, 1d, and 1e, were accepting other substrates with substituents. These selected as candidates for the activity testing of WT results indicated that the optimal substrate of EcMAL and several representative mutants. Notably, there was appears at each stage of evolutionary route and the pos- trace or even no specific activity observed for the WT. sibility of developing in different catalytic directions. Moreover, compared with the bulky caffeic acid, dif - ferent mutants exhibited obviously different catalytic Structure and computational simulation analysis of EcMAL activities (Ni et  al. 2020). Interestingly, the triple site variants mutant M6 showed significant improvement in activity To gain insight into the structure–activity relationship of toward 1b and was even better than M8 (Fig.  3). The the dramatically altered substrate specificity, the three- M8 mutant also obviously shows a great advantage in dimensional structures of EcMAL and EcMAL were WT M8 first compared by Yasara. As shown in Fig.  4, three polar mutant residues near the active center were replaced with non-polar residues (Q172R, Q329F, C361L) in EcM AL , M8 which increased the hydrophobic interaction between the active site and the substrate. Moreover, the residues H194, Q329 and K331 form the catalytic triad (Teze et al. 2020). Among them, the E329 responsible for stabiliz- ing the enolate anion was replaced with phenylalanine, which increased the pi–pi stacked interaction with the substrate. Because the catalytic active center of EcMAL is a wide crack shape, there is theoretically no situation in which the substrate has difficulty in contacting the active center because of the spatial resistance. Therefore, it is speculated that the critical factor affecting the enzyme catalytic activity is the unstable binding between the sub- strate and enzyme. The docking of the mutant and the substrate also confirmed this view, as the catalytic pocket of the enzyme was more accessible to the substrate. To investigate the conformational changes of the cata- lytic active site, whole systems were equilibrated by MD at 10  ns without any restriction (Teze et  al. 2020). Our Fig. 3 Activity fingerprints of Ec MAL and its variants (M6–M8) with goal is to identify conformational changes that regulate different substrates. The activity was measured by HPLC, and relative the accessibility of the active sites. Based on the snap- activity is measured as a percentage of the optimum activity of each shots of the MD simulations, it is easy to observe that substrate (1a caffeic acid, 1b cinnamic acid, 1c p‑hydroxycinnamic because of the substitution of phenylalanine at position acid, 1d methylcinnamic acid, 1e acrylic acid) Fig. 4 Comparison of hydrogen bonding in EcMAL (A) and its variant EcMAL (B). The docking substrate (1a) and amino acid residues are WT M8 shown in sticks. The hydrogen bonds are shown in black imaginary lines N i et al. Bioresour. Bioprocess. (2021) 8:103 Page 7 of 9 329, the enhancement in electrostatic attraction to aro- Table 3 Kinetic characterization of the EcMAL variants M6–M8 to substrates 1a and 1b matic substrates makes it more accessible to the bind- ing domain. The average per residue root mean square −1 Enzyme Substrate K (mM) K (min ) K /K m cat cat m −1 −1 fluctuation (RMSF) of the Cα atoms of a monomer in and (min  mM ) mutants EcMAL was calculated as a measurement of protein flexibility (Additional file  1: Figure S3). Residues L384 M8 1a 1.32 ± 0.09 61.76 ± 0.8 46.8 and M389 near the tunnel in the EcMAL exhibited WT M7 1a 1.25 ± 0.05 46.02 ± 0.4 36.8 lower flexibility, which was unfavorable to the bulky M6 1a 1.47 ± 0.05 34.5 ± 0.74 23.5 substrate in and out of the tunnel. The RMSF changes M8 1b 1.34 ± 0.06 31.48 ± 0.63 23.5 of the mutant residues also supported this hypothesis. M7 1b 1.96 ± 0.08 21.5 ± 0.38 11.0 M6 1b 1.23 ± 0.03 42.5 ± 0.72 34.6 Three parallels were measured for each sample. The reaction mixture contained Catalytic performance and substrate specificity of Ec mal 2+ 500 mM Tris–HCl buffer (pH 8.5), 500 mM NH Cl, 20 mM Mg , 1a: caffeic acid (0.25–10 mM)/1b: cinnamic acid (0.2–10 mM) variants We subsequently determined the catalytic perfor- mance of EcMAL variants for L-dopa synthesis. The showed strong activity with the lowest Km (1.23  mM) engineered EcMALs were used to react under optimal observed. This may be due to the different epistatic conditions. As shown in Fig.  5, the hydroamination effects of different residues, which simultaneous addition reaction catalyzed by variant M8 reached a mutant near the active center and channel of EcMAL, conversion rate higher than 50% within 12  h. Mean- resulting in different non-covalent forces on different while, it was also significantly improved compared substrates. to other mutants. To further evaluate the specificity responsible for different substrates in EcMAL vari - ants, the kinetic parameters of M6–M8 with substrates Conclusion 1a–1b were also considered (Table 3). M8 was obtained In summary, with the aim of activating EcMAL for the from M7 through the iterative mutation of two resi- amination of bulky unsaturated carboxylic acids, we dues 73 and 389. However, it is economical to achieve applied structure-guided strategies for the first time a large increase in the turnover rate (Kcat from 36.8 to simultaneously perform saturation mutagenesis on to 46.8) with a small sacrifice of affinity (Km from 1.25 the positions of the residues lining the binding pocket to 1.32  mM) for substrate 1a. Instead, the best variant and surrounding the substrate tunnel to successfully for 1a did not exert higher activity toward 1b, which evolve the enzyme. Iterative saturation mutagenesis has was equipped with a relatively smaller substituent. M6 been applied to optimize the results. Using an effective solid-phase screening method based on the oxidative color-forming reaction of polyphenols by tyrosinase, the effective variants could be quickly screened. Com - pared with traditional chromatographic screening, this method significantly improves the screening efficiency of the mutant libraries. After several rounds of screening, a series of variants with pronounced increases in catalytic activity and stereoselectivity for bulky substrates (includ- ing caffeic acid) were obtained, achieving a substrate specificity that shifted from small to large. Subsequently, the representative mutants were selected to explore the adaptability of bulky substrates by accepting four differ - ent substituted aromatic unsaturated carboxylic acids. Meanwhile, the synthesis of levodopa, an anti-dementia drug, was further investigated. The activity of the optimal mutant (M8) to caffeic acid increased by about 15-fold, and the conversion rate was more than 50% within 12 h. Fig. 5 Progress curves of caffeic acid catalyzed by the purified To gain deeper insight, further study into the crucial resi- enzymes of three EcMAL variants (M6, M7, M8). The enzymatic amination addition of caffeic acid (10 mM) was performed at 30 °C dues of engineered enzymes was carried out to explore and pH 8.5 with a dose of each 0.5 mg/mL purified enzymes under the regulation mechanism in the substrate spectrum. MD the same conditions simulation analysis and tunnel prediction were used to Ni et al. Bioresour. Bioprocess. (2021) 8:103 Page 8 of 9 from Aspergillus oryzae. Nat Chem 9:961–969. https:// doi. org/ 10. 1038/ explore the binding relationship between the substrate nchem. 2782 and enzyme in bulky substrate catalysis. We look forward Bea HS, Park HJ, Lee SH, Yun H (2011) Kinetic resolution of aromatic β‑amino to using this work as a starting point to develop more acids by ω‑transaminase. Chem Commun 47:5894–5896. https:// doi. org/ 10. 1039/ c1cc1 1528f valuable enzymatic hydroamination addition reactions Cheng X, Chen X, Feng J, Wu Q, Zhu D (2018) Structure‑ guided engineering to achieve green and economic synthesis of unnatural of: meso‑ diaminopimelate dehydrogenase for enantioselective reductive amino acids. amination of sterically bulky 2‑keto acids. Catal Sci Technol 8:4994–5002. https:// doi. org/ 10. 1039/ c8cy0 1426d De Raffele D, Martí S, Moliner V (2020) Understanding the directed evolution of de novo Retro‑Aldolases from QM/MM studies. ACS Catal 10:7871–7883. Abbreviations https:// doi. org/ 10. 1021/ acsca tal. 0c011 65 MAL: Methylaspartate lyase; EcMAL: Methylaspartate lyase from Escherichia coli Fibriansah G, Veetil VP, Poelarends GJ, Thunnissen AMWH (2011) Structural O157:H7; AL: Ammonia lyase; l ‑ dopa: Levodopa; DMSO: Dimethyl sulfoxide; basis for the catalytic mechanism of aspartate ammonia lyase. Biochemis‑ IPTG: Isopropyl‑β‑d‑thiogalactoside; MD: Molecular dynamics; RMSF: Root try 50:6053–6062. https:// doi. org/ 10. 1021/ bi200 497y mean square fluctuation. Fordjour E, Adipah FK, Zhou S, Du G, Zhou J (2019) Metabolic engineering of Escherichia coli BL21 (DE3) for de novo production of l ‑DOPA from Supplementary Information d ‑ glucose. Microb Cell Fact. https:// doi. org/ 10. 1186/ s12934‑ 019‑ 1122‑0 Heath RS, Pontini M, Bechi B, Turner NJ (2014) Development of an R‑selective The online version contains supplementary material available at https:// doi. amine oxidase with broad substrate specificity and high enantioselectiv‑ org/ 10. 1186/ s40643‑ 021‑ 00456‑5. ity. ChemCatChem 6:996–1002. https:// doi. org/ 10. 1002/ cctc. 20130 1008 Hu Y, Xu W, Hui C, Xu J, Huang M, Lin X, Wu Q (2020) The mutagenesis of a Additional file 1: Scheme S1. Asymmetric amination of caffeic acid by single site for enhancing or reversing the enantio‑ or regiopreference EcMAL for the production of L‑ dopa. Figure S1. Primer design and library of cyclohexanone monooxygenases. Chem Commun 56:9356–9359. creation of WT Ec‑MAL. Figure S2. Schematic representation of a solid‑https:// doi. org/ 10. 1039/ d0cc0 3721d phase screening assay. Figure S3. Molecular dynamics simulation results Kramer L, Le X, Hankore ED, Wilson MA, Guo J, Niu W (2019) Engineering of EcMAL. Table S1. List of primers for mutation library construction. and characterization of hybrid carboxylic acid reductases. J Biotechnol 304:52–56. https:// doi. org/ 10. 1016/j. jbiot ec. 2019. 08. 008 Lambrughi M, Maršić ŽS, Saez‑ Jimenez V, Mapelli V, Olsson L, Papaleo E (2019) Acknowledgements Conformational gating in ammonia lyases. Biorxiv. https:// doi. org/ 10. We thank Rosalie Tran, Ph.D., from Liwen Bianji (Edanz) (www. liwen bianji. cn/), 1101/ 583088 for editing the English text of a draft of this manuscript. Leese C, Fotheringham I, Escalettes F, Speight R, Grogan G (2013) Cloning, expression, characterisation and mutational analysis of l ‑aspartate Authors’ contributions oxidase from Pseudomonas putida. J Mol Catal B Enzym 85–86:17–22. Conceived and designed the experiments: WYL Performed the experiments: https:// doi. org/ 10. 1016/j. molca tb. 2012. 07. 008 ZFN, PX. Analyzed the data: ZFN. Contributed reagents/materials/analysis Li A, Qu G, Sun Z, Reetz MT (2019) Statistical analysis of the benefits of focused tools: MHZ, WYL. Wrote the paper: ZFN, WYL. All authors read and approved saturation mutagenesis in directed evolution based on reduced amino the final manuscript. acid alphabets. ACS Catal 9:7769–7778. https:// doi. org/ 10. 1021/ acsca tal. 9b025 48 Funding Li R, Wijma HJ, Song L, Cui Y, Otzen M, Tian Y, Du J, Li T, Niu D, Chen Y, Feng J, Funding was provided by the National Natural Science Foundation of China Han J, Chen H, Tao Y, Janssen DB, Wu B (2018) Computational redesign (21908070, 21878105), the National Key Research and Development Program of enzymes for regio‑ and enantioselective hydroamination article. Nat of China (2018YFC1603400), the Key Research and Development Program of Chem Biol. https:// doi. org/ 10. 1038/ s41589‑ 018‑ 0053‑0 Guangdong Province (No. 2019B020213001). Liu B, Qu G, Li JK, Fan W, Ma JA, Xu Y, Nie Y, Sun Z (2019) Conformational dynamics‑ guided loop engineering of an alcohol dehydrogenase: cap‑ Availability of data and materials ture, turnover and enantioselective transformation of difficult ‑to ‑reduce All data generated or analyzed during this study are included in this article. ketones. Adv Synth Catal 361:3182–3190. https:// doi. org/ 10. 1002/ adsc. 20190 0249 Liu Y, Xu G, Zhou J, Ni J, Zhang L, Hou X, Yin D, Rao Y, Zhao Y‑L, Ni Y (2020) Declarations Structure‑ guided engineering of d ‑ carbamoylase reveals a key loop at substrate entrance tunnel. ACS Catal 10:12393–12402. https:// doi. org/ 10. Ethics approval and consent to participate 1021/ acsca tal. 0c029 42 Not applicable. 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Structure-guided protein engineering of ammonia lyase for efficient synthesis of sterically bulky unnatural amino acids

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Abstract

Enzymatic asymmetric amination addition is seen as a promising approach for synthesizing amine derivatives, espe‑ cially unnatural amino acids, which are valuable precursors to fine chemicals and drugs. Despite the broad substrate spectrum of methylaspartate lyase (MAL), some bulky substrates, such as caffeic acid, cannot be effectively accepted. Herein, we report a group of variants structurally derived from Escherichia coli O157:H7 MAL (EcMAL). A combined mutagenesis strategy was used to simultaneously redesign the key residues of the entrance tunnel and binding pocket to explore the possibility of accepting bulky substrates with potential application to chiral drug synthesis. Libraries of residues capable of lining the active center of EcMAL were then constructed and screened by an effec‑ tive activity solid‑phase color screening method using tyrosinase as a cascade catalyst system. Activity assays and molecular dynamics studies of the resultant variants showed that the substrate specificity of Ec MAL was modified by adjusting the polarity of the binding pocket and the degree of flexibility of the entrance tunnel. Compared to M3, the optimal variant M8 was obtained with a 15‑fold increase in catalytic activity. This structure ‑based protein engineering of EcMAL can be used to open new application directions or to develop practical multi‑ enzymatic processes for the production of various useful compounds. Keywords: Biocatalysis, Methyl aspartate lyase, Substrate specificity, Protein engineering, Solid‑phase color screening Introduction of unnatural amino acids, having prominent advantages The hydroamination of olefins and carbon–nitrogen of economy and sustainability (Zhang et  al. 2020a). Six bond-forming reactions of unsaturated carboxylic acids classes of enzymes have been applied for this purpose, offer a vast range of applications in the synthesis of fine as exemplified by ω-transaminase (Mathew et  al. 2017; chemicals (Raj et  al. 2012). However, under classical Bea et  al. 2011), nitrilase (Yu et  al. 2019), amino acid conditions, the catalysts used are potentially hazard- dehydrogenase (Zhang et  al. 2015), ammonia lyase (AL) ous to the environment. With increasing emphasis on (Zhang et  al. 2020b), lipase (Zeng et  al. 2018), and tau- the concept of green, clean, and sustainable chemistry, tomerase (Liu et al. 2020). However, based on the princi- alternative strategies, such as biological methods, have ples of atomic-economy and cost-effectiveness, the most been proposed for implementing such reactions (Wu straightforward approach is the use of AL as the catalyst et  al. 2020). Enzymatic transformation of unsaturated to drive the hydroamination of unsaturated carboxylates carboxylic acids is an ideal approach for the production (Fibriansah et  al. 2011). This mild biocatalytic process would offer a welcome alternative for the synthesis of unnatural amino acids and their derivatives. ALs, belonging to the enolase family, specifically *Correspondence: wylou@scut.edu.cn Laboratory of Applied Biocatalysis, School of Food Science use unsaturated carboxylates as substrates for the and Engineering, South China University of Technology, No. 381 Wushan hydroamination reaction to generate unnatural amino Road, Guangzhou 510640, Guangdong, China © The Author(s) 2021. 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/. Ni et al. Bioresour. Bioprocess. (2021) 8:103 Page 2 of 9 acids and their derivatives (Turner 2011). Among Materials and methods ALs, MAL has attracted much attention because of its Chemicals broad substrate specificity and its acceptance of a vari- PrimerSTAR max DNA polymerase was obtained from ety of amines (Leese et  al. 2013). Recently, MAL and TaKaRa Biotechnology Co. (Dalian, China). Escherichia its variants have been reported to catalyze C–N bond coli strains BL21 (DE3) and DH5α were bought from formation in the synthesis of artificial dipeptide sweet- TransGen Biotech Co., Ltd. (Beijing, China). All other eners, showing that they possess broad amine scope in commercial chemicals were of analytical grade or above, accepting unnatural substrates (Zhang et  al. 2020b). and were purchased from Aladdin (Shanghai, China), However, despite these notable advances, unnatural Macklin (Shanghai, China), TCI (Tokyo, Japan) or Sigma- amino acids based on large-frame organic acids remain Aldrich (St. Louis, USA). largely unstudied. They can be envisaged as playing an important role in the synthesis of many chemicals, Homology modeling and molecular docking such as 3,4-dihydroxycinnamic acid, a precursor in the The EcMAL homology model was created using SWISS- production of levodopa (l -dopa) (Fordjour et al. 2019). MODEL (https:// swiss model. expasy. org/), taking MAL The potential of enzymes to be modified by directed from Clostridium tetanomorphum (PDB ID:1KKR) as a evolution has attracted much attention in recent years template (Lambrughi et  al. 2019). The modeled protein (Kramer et  al. 2019). Structure-guided protein engi- structure was evaluated through a Ramachandran plot, neering has replaced the natural mutation of enzymes and the percentage of residues in the allowed region was as one of the main tools of evolution (Cheng et  al. 98.6%. Molecular docking was performed with Yasara 2018; Renata et  al. 2015). For example, Wu and co- using the default program parameters. The center coor - workers used structure-based computational enzyme dinates of the box were determined by visual molecu- design to successfully convert aspartase into a series of lar dynamics, and the box size was set as 20  Å in each complementary hydroamination biocatalysts showing dimension. The docking results were selected according excellent regio- and enantioselectivity (Li et  al. 2018). to binding affinities and molecular conformations (Cheng However, the direction of directed evolution is uncon- et  al. 2018). PyMOL was used to display and analyze the trollable, so the correct selection of the mutagenesis modeled protein (Yuan et al. 2016). Amino acids with low strategies and the amount of screening verification conservation score at the entrance of the active pocket work are key in determining the ultimate degree of were chosen as hotspots (Yu et  al. 2019). CAVER 3.0 success (Zhou et al. 2016). Screening is the bottleneck (http:// www. caver. cz) was used to identify the tunnels of directed evolution, and two strategies are presently present in EcMAL (Zhang et al. 2019). in use. In the first, the focus is on developing efficient mutagenesis strategies to generate high-quality librar- PCR‑based methods for site mutagenesis and library ies to minimize the detection workload (Zhou et  al. construction of EcMAL 2016; Hu et al. 2020; Li et al. 2019). In the second, the Site-directed mutagenesis and combinatorial active site aim is to establish high-throughput screening meth- saturation testing were conducted by the overlap PCR ods, allowing rapid discrimination based on colorimet- and megaprimer approach with PrimerSTAR max DNA ric or optical changes (Li et al. 2019, 2020). polymerase. The reaction system had a total volume of 50 Our previous studies have already led to the mining μL, comprising 25 μL of PrimerSTAR max polymerase, of an MAL from E. coli O157:H7, which was found to 0.5 μL (50–100 ng) of template DNA, and 200 μm prim- catalyze the hydroamination of short-chain unsatu- ers mix (2 μL each), made up to the specified volume with rated acids such as fumaric acid or itaconic acid to the water. PCR amplification protocol for short fragments: corresponding amino acids with good selectivity. How- 98 °C for 2 min; (98 °C for 30 s, 55 °C for 30 s, 72 °C for ever, a dramatic decrease in reactivity was observed 1  min) × 30 cycles; 72  °C for 5  min. PCR products with when the substrate side chains increased. Therefore, the above short fragments were then used as primers using structural information as a guide to expand for mega-PCR: 98 °C for 2 min; (98 °C for 30 s, 60 °C for the substrate scope of engineering MALs is the main 30  s, 72  °C for 7  min) × 30 cycles; 72  °C for 10  min. The objective. In this study, we aimed to probe new resi- PCR products were resolved by agarose gel electropho- dues located near the active center and entrance tun- resis and purified using a Sangon Biotech purification nel, which may affect the acceptance of bulky aromatic kit. Digestion reaction program: 2 μL of NEB CutSmart unsaturated acids. We then further adjusted them to buffer and 1 μL of Dpn I were added for every 20 μL of expand the substrate spectrum of MAL. identified PCR product, and the mixture was incubated for 4  h at 37  °C. After thermal inactivation at 80  °C for N i et al. Bioresour. Bioprocess. (2021) 8:103 Page 3 of 9 20  min, plasmids containing the mutated gene were washed twice with 0.9% NaCl solution. The cells were directly transformed into E. coli BL21 (DE3) and then resuspended in buffer A (200  mM Tris–HCl buffer con - plated on a Luria–Bertani (LB) agar plate with 100 μg/mL taining 20  mM imidazole, 500  mM NaCl, pH 8.5) and of ampicillin. disrupted by ultrasonication. The cell lysates were cen - Multi-site simultaneous mutagenesis is based on spe- trifuged at 10,000g for 10  min at 4  °C to remove insolu- cific amino acid selection and primer design, which ble debris. The protein supernatant samples were loaded determines the size and quality of the constructed library. onto a HisTrap Ni–NTA FF column (GE Healthcare, Here, the 19 residues involved in EcMAL were divided USA) and eluted with buffer B (in which the imidazole into five groups (A–E). The short fragments between concentration was increased to 250  mM). The purified multiple mutation sites, as primers for the next round proteins were then further desalted by passage through a of amplification reactions, were then amplified by PCR desalination column (GE Healthcare, USA). The Bradford (Wang et  al. 2016). The purified short fragments were method was used to determine the protein concentration. used as primers to amplify the whole plasmid PET32a- EcMAL, leading to the final range of plasmids for library Specific activity and enantioselectivity assays generation. After digestion by Dpn I, the PCR products of the wild‑type and mutant enzymes were transformed into E. coli BL21 (DE3) cells to cre- The kinetic parameters of EcMAL were first measured ate a library for screening. Applying standard transfor- with varying concentrations of 1a (0.25–10 mM) and 1b mation procedures, the transformants were spread on (0.2–10  mM) as substrates by detecting the initial reac- HyBond-N membranes, which were then placed on the tion rate of the protein and curve-fitting according to the surface of LB/ampicillin (Amp, 100  μg/mL) agar plates. Michaelis–Menten equation. The specific activities and After incubation overnight at 37  °C, the membranes stereoselectivities of the EcMAL mutants toward 1a, 1b, were transferred to new LB/amp plates with 0.1  mM and 1c were then measured using purified enzymes. The isopropyl-β-d-thiogalactopyranoside (IPTG) and incu - reactions were carried out as follows: in a reaction sys- bated for 12 h at 30 °C. Each membrane was then trans- tem with a total volume of 2  mL, substrate 1a (10  mM), ferred to a clean plate and stored at – 20 °C prior to use. 1b (10 mM), or 1c (5 mM) in dimethyl sulfoxide (0.5 mL) and the purified enzyme were mixed with Tris–HCl Screening procedures (500  mM, pH 8.5). The mixture was shaken at 180  rpm Our solid-phase screening procedure is based on the and 30  °C, during which samples were withdrawn at analysis method devised by Turner’s group (Aleku et  al. regular intervals and extracted with an equal volume of 2017). Membranes containing the clones were repeat- ethyl acetate. The progress of the reaction was monitored edly frozen in liquid N and thawed four times to lyse the by HPLC analysis and all experiments were conducted in cells. The membranes were then placed on top of a filter triplicate. paper that was previously loaded with final concentra - 2+ tions of 10  mM screening substrate, 20  mM Mg , and Production of LOPA 500 mM NH Cl in 500  mM Tris–HCl buffer (pH 8.5). In a mixed system with a reaction volume of 20  mL, After incubation for 6 h at 30 °C, color-changed colonies the final concentrations of the components were as fol - were picked and inoculated into LB/amp for subsequent lows: 10 mM caffeic acid (with 2% (v/v) DMSO), 20 mM activity detection and mutant sequencing comparison. MgCl , 500  mM NH Cl, 200  mg/L purified enzyme, and 2 4 The number of mutants screened is calculated according 200  mM Tris–HCl buffer (pH 8.5). The reaction flask to the 95% mutation coverage determined by mutation was shaken at 180 rpm and 30 °C. Samples were regularly toolbox developed by Reezt’s team (Reetz et al. 2005). withdrawn and analyzed by HPLC. Protein expression and purification Results and discussion Wild-type and positive variants of EcMAL were inocu- Selection and grouping of key amino acids of Ecmal lated in 20  mL of LB medium containing 100  μg/mL of Based on our previous studies, we found that EcMAL ampicillin and incubated at 37 °C and 180 rpm. The over - could accept a range of short-chain unsaturated carbox- night cultures were transferred into 500  mL of medium ylic acids, including fumaric acid, mesaconic acid, and at a 1% inoculum level. When the absorbance of the itaconic acid, for the asymmetric synthesis of unnatural medium at 600 nm (OD600) reached 0.6–0.8, a final con - amino acids (Ni et  al. 2020). However, EcMAL showed centration of 0.1  mM IPTG was added, and the cultiva- low activity in the catalysis of bulky unsaturated car- tion temperature was adjusted to 16 °C to induce protein boxylic acids. This difference prompted us to investigate overexpression. After 20 h, each culture was harvested by the catalytic mechanism of EcMAL from a structural centrifuging at 8000g for 5  min, and the precipitate was perspective, with a view to further improve the catalytic Ni et al. Bioresour. Bioprocess. (2021) 8:103 Page 4 of 9 activity and stereoselectivity toward bulky substrates. We of degenerate NNK codons (N = A, G, C, T; K = G) for chose caffeic acid (1a) as the model compound for assess - saturation mutation would fully cover all possible amino ing the activity of EcMAL mutants, the basic reaction of acids, it is also accompanied by a dramatic increase in the which is shown in Additional file  1: Scheme S1. Four aro- number of screening (Wittmann et al. 2020). The degen - matic unsaturated carboxylic acids (cinnamic acid (1b), erate NDT codons (D = A, G, T) encoding the 12 differ - p-hydroxycinnamic acid (1c), methylcinnamic acid (1d), ent types of amino acids (C/F/H/D/N/S/I/G/V/L/Y/R) and acrylic acid (1e)) with different substituents were better meet the requirements of a “smart” library (Reetz also selected. These bulky compounds were selected as et  al. 2006). Thus, NDT was considered as a simplified substrates because their amination products are impor- codon for library construction. Because the combina- tant components or building blocks of therapeutic drugs, tion of simultaneous mutations increases exponentially, such as the Parkinson’s curative l-dopa. Considering that it is still necessary to screen at least 10 levels to achieve polyphenols can undergo a rapid color-forming reac- the theoretical 95% coverage (Wang et al. 2017). On this tion under the action of tyrosinase, we selected 1a as the basis, we developed a solid-phase screening method that screening substrate to assess the efficacies of the enzyme could rapidly screen 10 level transformants through variants. Based on the homologous three-dimensional color changes on a single plate. The single colonies that model of EcMAL, Yasara and CAVER 3.0 were used for met the requirements were subsequently selected for fur- molecular docking and substrate tunnel identification, ther activity testing (Tang et al. 2018). respectively (Liu et al. 2019; Heath et al. 2014). The sub - strate-binding pocket and substrate tunnel have been Screening and identification of active Ec mal variants demonstrated to have varying degrees of impact on the First of all, we designed mutation primers according to properties of the enzyme, thereby enhancing the differen - different mutation groups, and constructed the mutant tiation of directed evolution (De Raffele et  al. 2020). We libraries after amplification (Additional file  1: Figure simultaneously considered two groups of residues, those S1). Using the solid-phase color screening procedure lining the binding pocket (73, 170, 172, 194, 329, 384) and described in the experimental part, we performed high- those surrounding the substrate tunnel (196, 198, 199, throughput screening of five mutant libraries (Additional 240, 277, 307, 308, 356, 365, 389). Three residues (331, file  1: Figure S2). In the first round of screening, several 360, 361) participating in both regions were also taken mutants with activity to substrate 1a were screened from into account (Fig. 1). libraries A to E, and the location and type of mutations According to the type and spatial location of the 19 were further determined by sequencing. The screening selected residues, we attempted to divide them into five results are listed in Table  2 (M1–M7). Clearly, although groups, as shown in Fig. 2 and Table 1. Although the use the double mutations (M1–M3) showed new trace Fig. 1 EcMAL residues chosen for saturation mutagenesis marked in the homology model built by the crystal structure of MAL (PDB: 1KKR). A Active site mutation sites (yellow) selected on the basis of induced fit docking of amine 1 (green). B Residues surrounding the substrate access tunnel likewise chosen for mutagenesis (blue). The above residues were obtained through the molecular docking of Yasara and the channel recognition function of CAVER 3.0, respectively N i et al. Bioresour. Bioprocess. (2021) 8:103 Page 5 of 9 Fig. 2 Spatial structure information of substrate–enzyme docking. A The position relationship between EcMAL channels and the active sites. The target residues of libraries A–E are displayed in purple sticks. The substrate tunnel is shown in yellow and blue. B Poses of caffeic acid docked in the modeled structure of EcMAL. The substrate caffeic acid is shown in azure and the selected residues envelop the substrate in a surface view. The visualization of EcMAL channel, substrate and target amino acid residues was achieved by Pymol and Yasara Table 2 Properties of the EcMAL variants Table 1 Grouping of the 19 chosen EcMAL residues into five randomization sites (A, B, C, D and E) and the NDT codes used in Enzyme and mutants Mutation sites Specific % ee saturation mutagenesis activity (U/ g ) protein Randomization site Code c c WT EcMAL None nd nd M1 Q329S/K331R 5.4 ± 0.5 16 (S) A: 170, 172, 198, 199 NDT M2 Q73F/Y240C 4.4 ± 0.4 4.4 (S) B: 194, 196, 384, 389 NDT M3 F170L/E172F 2.5 ± 0.5 39 (S) M4 Q329S/K331G/C361C 6.2 ± 0.8 86(S) C: 307, 308, 356, 360 NDT M5 Q73V/Y240C/D277C 7.3 ± 0.1 28(S) M6 F170S/E172R/D307L 9.0 ± 0.3 79 (S) D: 329, 331, 361, 365 NDT M7 Q329F/K331R/Y356S/ 26.2 ± 0.2 99 (S) C361L E: 73, 240, 277 NDT M8 E172R/Y240S/D307L/ 38.6 ± 0.5 99 (S) Q329F/Y356S/C361L The red numbers denote the “active site” positions, the green ones as the “tunnel” positions, and blue ones as the “shared” positions Specific activity was determined at pH 8.0 and 30 °C using purified enzyme and omeprazole sulfide The ee values were determined by HPLC activity toward caffeic acid compared with the wild type, Not detected its activity and stereoselectivity were far from the expec- The bold mutations indicate newly involved mutation(s) in each round tations (Li et  al. 2019). However, the coincidence that the effective mutants were concentrated in libraries B, D, and E caught our attention. The triple mutants (M4– and confirming the specific activities, we identified the M6) screened by these libraries further indicated that the best variants (M8) toward the model substrates, which selected hot sites in libraries B, D, and E had a great influ - are shown in Table  2. Compared with the catalytic per- ence on the enzyme to accept bulky substrates. In par- formance identified by other mutants, the M8 presented ticular, a quadruple mutation M7 (Q329F/K331R/Y356S/ about 15-fold improvement and the selectivity was also C361L) suddenly exhibited a mutation transition effect, greatly improved. Considering that M8 was created by with remarkable improvements in catalytic activity and partial iterative mutation of M7, and the catalytic activity selectivity. Motivated by these results, M7 was chosen as was improved while the stereoselectivity was maintained, the parental enzyme for further iterative combinations. the results indicate a prominent synergistic influence Subsequently, a second round of screening experiments among these six residues. were implemented for libraries B and E using M7 as the To fully investigate the catalytic performance of the template. After screening approximately 5000 clones selected mutants, several substrates with varying sizes Ni et al. Bioresour. Bioprocess. (2021) 8:103 Page 6 of 9 and structures, including 1a, 1b, 1c, 1d, and 1e, were accepting other substrates with substituents. These selected as candidates for the activity testing of WT results indicated that the optimal substrate of EcMAL and several representative mutants. Notably, there was appears at each stage of evolutionary route and the pos- trace or even no specific activity observed for the WT. sibility of developing in different catalytic directions. Moreover, compared with the bulky caffeic acid, dif - ferent mutants exhibited obviously different catalytic Structure and computational simulation analysis of EcMAL activities (Ni et  al. 2020). Interestingly, the triple site variants mutant M6 showed significant improvement in activity To gain insight into the structure–activity relationship of toward 1b and was even better than M8 (Fig.  3). The the dramatically altered substrate specificity, the three- M8 mutant also obviously shows a great advantage in dimensional structures of EcMAL and EcMAL were WT M8 first compared by Yasara. As shown in Fig.  4, three polar mutant residues near the active center were replaced with non-polar residues (Q172R, Q329F, C361L) in EcM AL , M8 which increased the hydrophobic interaction between the active site and the substrate. Moreover, the residues H194, Q329 and K331 form the catalytic triad (Teze et al. 2020). Among them, the E329 responsible for stabiliz- ing the enolate anion was replaced with phenylalanine, which increased the pi–pi stacked interaction with the substrate. Because the catalytic active center of EcMAL is a wide crack shape, there is theoretically no situation in which the substrate has difficulty in contacting the active center because of the spatial resistance. Therefore, it is speculated that the critical factor affecting the enzyme catalytic activity is the unstable binding between the sub- strate and enzyme. The docking of the mutant and the substrate also confirmed this view, as the catalytic pocket of the enzyme was more accessible to the substrate. To investigate the conformational changes of the cata- lytic active site, whole systems were equilibrated by MD at 10  ns without any restriction (Teze et  al. 2020). Our Fig. 3 Activity fingerprints of Ec MAL and its variants (M6–M8) with goal is to identify conformational changes that regulate different substrates. The activity was measured by HPLC, and relative the accessibility of the active sites. Based on the snap- activity is measured as a percentage of the optimum activity of each shots of the MD simulations, it is easy to observe that substrate (1a caffeic acid, 1b cinnamic acid, 1c p‑hydroxycinnamic because of the substitution of phenylalanine at position acid, 1d methylcinnamic acid, 1e acrylic acid) Fig. 4 Comparison of hydrogen bonding in EcMAL (A) and its variant EcMAL (B). The docking substrate (1a) and amino acid residues are WT M8 shown in sticks. The hydrogen bonds are shown in black imaginary lines N i et al. Bioresour. Bioprocess. (2021) 8:103 Page 7 of 9 329, the enhancement in electrostatic attraction to aro- Table 3 Kinetic characterization of the EcMAL variants M6–M8 to substrates 1a and 1b matic substrates makes it more accessible to the bind- ing domain. The average per residue root mean square −1 Enzyme Substrate K (mM) K (min ) K /K m cat cat m −1 −1 fluctuation (RMSF) of the Cα atoms of a monomer in and (min  mM ) mutants EcMAL was calculated as a measurement of protein flexibility (Additional file  1: Figure S3). Residues L384 M8 1a 1.32 ± 0.09 61.76 ± 0.8 46.8 and M389 near the tunnel in the EcMAL exhibited WT M7 1a 1.25 ± 0.05 46.02 ± 0.4 36.8 lower flexibility, which was unfavorable to the bulky M6 1a 1.47 ± 0.05 34.5 ± 0.74 23.5 substrate in and out of the tunnel. The RMSF changes M8 1b 1.34 ± 0.06 31.48 ± 0.63 23.5 of the mutant residues also supported this hypothesis. M7 1b 1.96 ± 0.08 21.5 ± 0.38 11.0 M6 1b 1.23 ± 0.03 42.5 ± 0.72 34.6 Three parallels were measured for each sample. The reaction mixture contained Catalytic performance and substrate specificity of Ec mal 2+ 500 mM Tris–HCl buffer (pH 8.5), 500 mM NH Cl, 20 mM Mg , 1a: caffeic acid (0.25–10 mM)/1b: cinnamic acid (0.2–10 mM) variants We subsequently determined the catalytic perfor- mance of EcMAL variants for L-dopa synthesis. The showed strong activity with the lowest Km (1.23  mM) engineered EcMALs were used to react under optimal observed. This may be due to the different epistatic conditions. As shown in Fig.  5, the hydroamination effects of different residues, which simultaneous addition reaction catalyzed by variant M8 reached a mutant near the active center and channel of EcMAL, conversion rate higher than 50% within 12  h. Mean- resulting in different non-covalent forces on different while, it was also significantly improved compared substrates. to other mutants. To further evaluate the specificity responsible for different substrates in EcMAL vari - ants, the kinetic parameters of M6–M8 with substrates Conclusion 1a–1b were also considered (Table 3). M8 was obtained In summary, with the aim of activating EcMAL for the from M7 through the iterative mutation of two resi- amination of bulky unsaturated carboxylic acids, we dues 73 and 389. However, it is economical to achieve applied structure-guided strategies for the first time a large increase in the turnover rate (Kcat from 36.8 to simultaneously perform saturation mutagenesis on to 46.8) with a small sacrifice of affinity (Km from 1.25 the positions of the residues lining the binding pocket to 1.32  mM) for substrate 1a. Instead, the best variant and surrounding the substrate tunnel to successfully for 1a did not exert higher activity toward 1b, which evolve the enzyme. Iterative saturation mutagenesis has was equipped with a relatively smaller substituent. M6 been applied to optimize the results. Using an effective solid-phase screening method based on the oxidative color-forming reaction of polyphenols by tyrosinase, the effective variants could be quickly screened. Com - pared with traditional chromatographic screening, this method significantly improves the screening efficiency of the mutant libraries. After several rounds of screening, a series of variants with pronounced increases in catalytic activity and stereoselectivity for bulky substrates (includ- ing caffeic acid) were obtained, achieving a substrate specificity that shifted from small to large. Subsequently, the representative mutants were selected to explore the adaptability of bulky substrates by accepting four differ - ent substituted aromatic unsaturated carboxylic acids. Meanwhile, the synthesis of levodopa, an anti-dementia drug, was further investigated. The activity of the optimal mutant (M8) to caffeic acid increased by about 15-fold, and the conversion rate was more than 50% within 12 h. Fig. 5 Progress curves of caffeic acid catalyzed by the purified To gain deeper insight, further study into the crucial resi- enzymes of three EcMAL variants (M6, M7, M8). The enzymatic amination addition of caffeic acid (10 mM) was performed at 30 °C dues of engineered enzymes was carried out to explore and pH 8.5 with a dose of each 0.5 mg/mL purified enzymes under the regulation mechanism in the substrate spectrum. MD the same conditions simulation analysis and tunnel prediction were used to Ni et al. Bioresour. Bioprocess. (2021) 8:103 Page 8 of 9 from Aspergillus oryzae. Nat Chem 9:961–969. https:// doi. org/ 10. 1038/ explore the binding relationship between the substrate nchem. 2782 and enzyme in bulky substrate catalysis. 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Journal

"Bioresources and Bioprocessing" – Springer Journals

Published: Oct 19, 2021

Keywords: Biocatalysis; Methyl aspartate lyase; Substrate specificity; Protein engineering; Solid-phase color screening

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