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Correlated mutations in hydroxysteroid dehydrogenases family

Correlated mutations in hydroxysteroid dehydrogenases family Background: Hydroxysteroid dehydrogenase enzymes belong to the short-chain dehydrogenase/reductase (SDR) superfamily and aldo-keto reductases (AKRs). SDR is involved in the metabolism of many compounds (hormones, lipids, etc.) and is present in almost all studied genomes. Two hundred members of hydroxysteroid dehydrogenases have been analysed in terms of natural mutational variability. The second superfamily comprises AKR superfamily group enzymes whose function is catalysing the oxidation and reduction of many substrates by binding NAD(P)H as a cofactor. This kind of study is the first approach for the hydroxysteroid dehydrogenase family. This information grants practical meaning to designing potential specific drugs to fight specific diseases caused by mutations. Methods: In the research, amino acid sequences of representatives of the hydroxysteroid dehydrogenase family were extracted from the UniProt database. In total, the analysed 200 sequences with the highest degree of similarity were shown by BLAST searches. In the sequence analyses, we used the following software: ClustalX (multiple sequence alignment), Consensus Constructor (creating consensus sequence), and CORM (finding correlated mutations). Results: The CORM program identified potential sites of correlated mutations in hydroxysteroid dehydrogenases. This program generated 18 tables of results that contain the amino acid positions of mutations. Seven of these are presented in this paper. Conclusions: The primary structure of the hydroxysteroid dehydrogenase family shows high variation. Keywords: aldo-keto reductases; correlated mutations; hydroxysteroid dehydrogenases; short-chain dehydrogenase/reductase. *Corresponding author: Agata yniewska, Department of Molecular Biology, Faculty of Biological Sciences, University of Zielona Góra, Zielona Góra, Poland, E-mail: agaw0606@gmail.com Jacek Leluk and Gabriela aroffe: Department of Molecular Biology, Faculty of Biological Sciences, University of Zielona Góra, Zielona Góra, Poland Hydroxysteroid dehydrogenases HSDs have multiple functions. They play an important role in the local production of steroid hormones in target tissues; they can transform active sex hormones into inactive forms, and vice versa. They also have the function of catalysing stereoselective reactions at a specific site so that all steroid hormones possess the appropriate isoform that either inactivates hormones or activates the ligand form. HSDs have oxidative and reductive properties so that their isoforms can act as molecular switches for `on' and `off' [1, 2]. SDR superfamily SDR is one of the largest superfamilies of proteins. These enzymes were identified as a separate and new group of oxidoreductases at the end of the year 1979; the term SDR was coined in 1991 [1, 3]. One of the characteristics of these enzymes is that their chains reach a length of about 250 amino acids. HSDs, which belong to this family, have a single binding domain of NAD(P)(H) and exhibit a wide variety of substrates [4, 5]. Another significant feature of this protein superfamily is the presence of characteristic sequence motifs that are arranged in a certain way. These motifs include the highly conserved triad of Ser, Tyr, and Lys of the active site and the nucleotide-binding Rossmann motif. The Rossmann motif consists of six to seven chains, which are surrounded by three to five -helical chains. SDR enzymes are involved in the metabolism of many compounds (hormones, lipids, etc.) and are present in almost all studied genomes (in humans, they are coded by >70 genes). The SDR superfamily is divided into five classes: classic, intermediate, extensive, diverse, and complex. They catalyse the major steps of inactivation or 18yniewska et al.: Mutations in HSDs activation in vitamins, steroids, prostaglandins, and other bioactive molecules by reduction and oxidation, respectively, in carbonyl and hydroxyl groups [2, 6] (Table 1). result was subjected to manual verification. The result of the ClustalX program ­ the multiple sequence alignment ­ was used in the CORM program to identify potential sites of correlated mutations and was further analysed in Consensus Constructor. AKR superfamily This is a family of enzymes whose function is catalysing the oxidation and reduction of many substrates by binding NAD(P)H as a cofactor. AKRs are oxidoreductases that share a common protein structure, /-barrels, which consist of eight -helices and eight -chains that are arranged inside the structure for rigidity. The structure of /-barrel contains the active site and pocket binding of NAD(P)H. AKR family enzymes have three big loops in the C-ends of barrels. Changes in this region of the enzyme determine the affinity of the substrates [9­11] (Table 2). Computer programs used ­ ClustalX: This program was used to match homologous sequences and create multiple sequence alignments. CORM: This program was used to search, analyse, and describe correlated mutations occurring in families of homologous proteins. Consensus Constructor: This program was used to create a consensus sequence from a set of matched sequences as well as a detailed analysis of these matches. Chimera: This program was used to create figures with three-dimensional structures. Methods Databases and the leader sequence In the research, the amino acid sequences of members of the HSD family were extracted from the UniProt database available on the Internet [14]. In the next step, the downloaded sequences were tested using BLAST if they show affinity to known members of the dehydrogenases family [15, 16]. The analysis used 200 sequences with the highest degree of similarity. The HSD analysis group adopted the sequence leader, which was based on database searches. The leader sequence was: 3 -HSD/Delta 54-isomerase type 2 (identification no. P26439). Homologous sequences were arranged using the ClustalX program [17]. The multiple sequence alignment Table 1:Characteristics of the superfamily: SDR [7, 8]. Build Main structural motif Cofactor Binding site Multimeric (monomer 25 kDa) Rossmann motif (-)2 NAD(P)(H) Motif Tyr-X-X-X-Lys Results The CORM program identified potential sites of correlated mutations in HSDs. This program generated 18 tables of results. Seven of them are presented in this paper. Table 3 shows the number of possible consequences of amino acid changes at position 97 concerning cysteine (C) or tyrosine (Y). A change of an amino acid in this Table 3:First cluster of correlated mutations in the HSD family. Position Amino Counts 117: -ACEFHKMQRVWY 128: -FGILMVY acid 97 97 C Y 101 81 E ACFHKMQRWY LM FIVY Table 4:Second cluster of correlated mutations in the HSD family. Position 138 138 Amino acid ­ E Counts 79 73 137: -EGHIKLPQRSTV ­ EIKLQRV Table 2:Characteristics of the superfamily: AKR [10, 12, 13]. Build Main structural motif Cofactor Binding site Monomeric (34 kDa) TIM-barrel structure (/)8 NAD(P)(H) Conservative tetrad: Tyr55, Asp50, Lys84, His117 Table 5:Third cluster of correlated mutations in the HSD family. Position 153 153 Amino acid ­ T Counts 93 73 149: -KLQRV ­ KLQR 154: -AEKLMQ ­ AEKLMQ yniewska et al.: Mutations in HSDs19 Table 6:Fourth cluster of correlated mutations in the HSD family. Position 154 154 Amino acid ­ K Counts 94 89 153: -AIPSTV -P AISTV Table 8:Sixth cluster of correlated mutations in the HSD family. Position Amino Counts 308: -DEHKLNQRTVY 331: -CFLPQRSWY acid 294 294 G Y 73 EHKLNQRTVY 83 -D CFLRW -PQ Table 7:Fifth cluster of correlated mutations in the HSD family. Position Amino Counts 230: -ACFILMSTV 387: -CEIKLMNPQRSTV acid 217 217 G S 78 CILMSTV 67 A CEKMNPRST -ILQV position to cysteine may result in the appearance of a mutation coupled at position 117 with original amino acids: ACEFHKMQRVWY and deletion marked by -. Subsequently, in this place can occur glutamic acid (E), and at position 128: -FGILMVY, where leucine (L) or methionine (M) may occur. However, regarding the occurrence at position 97, the amino acid tyrosine (Y) may result in the occurrence of amino acid changes at position 117, -ACEFHKMQRVWY on ACFHKMQRWY, and the position 128: -FGILMVY on FIVY. Table 4 shows the correlated mutations associated with amino acid changes at position 138 of the enzyme. At this position may occur 138 amino acid deletions, the result of which will be the next amino acid deletion at position 137. If, however, this place is substituted with glutamic acid (138), mutations may occur in position 137, which will change the primary amino acid -EGHIKLPQRSTV to another: EIKLQRV. The probability of a correlated mutation is also found in the deletion or change to an amino acid at position 153 of the proteins (see Table 5). The deletion at this place leads to the deletion of the following positions: 149 -KLQRV and 154 -AEKLMQ. A change of 153 amino acid to threonine (T) causes a further change to the amino acid at position 149, -KLQRV on KLQR, and at position 154: -AEKLMQ on any amino acid from the same set AEKLMQ. A mutation to an amino acid at position 154 (see Table 6) may take place in two ways. In the first case, the deletion of amino acid 154 as a result of mutations may cause another change in position 153: -AIPSTV on the deletion of this amino acid or its change to proline (P). In the second instance, the 154 amino acid can lead to mutations in the adjacent position 153: -AIPSTV on AISTV. Table 7 shows the next possible location of the mutation correlated in the HSD family. A mutation of the amino acid at position 217 determines the subsequent emergence of mutations at positions 230 and 387 (see Figure 1). The next result of the CORM program is shown in Table 8: the mutation of position 294 can cause the following mutations (see Figure 2). The consequence of mutational changes in position 381 in the HSD family is the emergence of several correlated mutations (see Table 9). A change of amino acid 381 to serine (S) will trigger further changes in the following positions: ­ 239: -AFILMV will change amino acid on isoleucine (I), leucine (L), or valine (V). ­ 329: -ACHIQRSTV will change amino acid on alanine (A), isoleucine (I), or valine (V). Mutation at position 217 Change amino acid on glycine (G) Change amino acid on serine (S) Mutation at position 230:-ACFILMSTV Mutation at position 387:-CEIKLMNPQRSTV Amino acid deletion Change amino acid on CILMSTV Change amino acid on CEKMNPRST Change amino acid on A Change amino acid on ILQV Figure 1:Pathways of correlated mutations caused by amino acid mutation at position 217. 20yniewska et al.: Mutations in HSDs Change amino acid on glycine (G) Mutation at position 308:-DEHKLNQRTVY Mutation at position 331:-CFLPQRSWY Change amino acid on EHKLNQRTVY Change amino acid on CFLRW Mutation at position 294 Mutation at position 308:-DEHKLNQRTVY Change amino acid on tyrosine (Y) Mutation at position 331:-CFLPQRSWY Change amino acid on D Amino acid deletion Change amino acid on PQ Figure 2:Pathways of correlated mutations caused by amino acid mutation at position 294. Table 9:Seventh cluster of correlated mutations in the HSD family. Position 381 381 Amino acid S Y Counts 89 76 239: -AFILMV ILV FM 329: -ACHIQRSTV AIV HQRS 355: -AGMPS AG M 380: -ACILQSV ILV ACS 386: -ACFHILMPQSTVY HLPV CMSTY 427: -ACEFHILMNQTVY -ACLMQT HNY 461: -CFIMSTY -FMY ST ­ ­ ­ ­ ­ 355: -AGMPS will change amino acid on alanine (A) or glycine (G). 380: -ACILQSV will change amino acid on isoleucine (I), leucine (L), or valine (V). 386: -ACFHILMPQSTVY will change amino acid on histidine (H), leucine (L), proline (P), or valine (V). 427: -ACEFHILMNQTVY will change amino acid on A, C, L, M, Q, T, or the deletion. 461: -CFIMSTY will change amino acid on phenylalanine (F), methionine (M), tyrosine (Y), or the deletion. ­ ­ ­ ­ ­ ­ ­ Change of amino acid 381 to tyrosine (Y) will trigger further changes in positions, as follows: 239: -AFILMV will change amino acid on phenylalanine (F) or methionine (M). 329: -ACHIQRSTV will change amino acid on histidine (H), glutamine (Q), arginine (R), or serine (S). 355: -AGMPS will change amino acid on methionine (M). 380: -ACILQSV will change amino acid on alanine (A), cysteine, (C), or serine (S). 386: -ACFHILMPQSTVY will change amino acid on cysteine (C), methionine (M), serine (S), threonine (T), or tyrosine (Y). 427: -ACEFHILMNQTVY will change amino acid on histidine (H), asparagine (N), or tyrosine (Y). 461: -CFIMSTY will change amino acid on serine (S) or threonine (T). Figure 3:Consensus sequence. yniewska et al.: Mutations in HSDs21 Consensus sequence The analysed group of proteins created a list of about 200 sequences showing the closest similarity to the reference sequence. They were aligned by the ClustalX program. Further, a detailed verification of the matches was carried out manually. Then, they were used by the Consensus Constructor [18] program, which created a consensus sequence, a collective summary of the studied group of enzymes. The parameters for use were a minimum amino acid count of 66 and a maximum conservatism (%) of 80.0. In Figure 3 the black background shows a strong conservative place, the grey background shows a moderately conserved place, and the white background shows a high variability of the amino acid. Figure 4:Selected correlated mutations incorporated in Table 3 in the molecule of human 17-HSD type 1 (1ry0.pdb). Pink position ­ primary mutational change (proline-97); green position ­ correlated mutation with the primary mutation (histidine-117 and leucine-128). Figure 5:(A, B) Selected correlated mutations incorporated in Table 4 in the molecule of human 17-HSD type 1 (1ry0.pdb). Pink position ­ primary mutational change (leucine-138); green position ­ correlated mutation with the primary mutation (valine-137). Figure 6:(A, B) Selected correlated mutations incorporated in Table 5 in the molecule of human 17-HSD type 1 (1ry0.pdb). Pink position ­ primary mutational change (aspartic acid-153); green position ­ correlated mutation with the primary mutation (leucine-149 and valine-154). 22yniewska et al.: Mutations in HSDs Figure 7:(A, B) Selected correlated mutations incorporated in Table 6 in the molecule of human 17-HSD type 1 (1ry0.pdb). Pink position ­ primary mutational change (valine-154); green position ­ correlated mutation with the primary mutation (aspartic acid-153). Discussion In this study, we observed the correlated mutations in the HSD family. The occurrence of correlated mutations in 17-HSD type 1 in Table 3 (see Figure 4) may result in major changes in the functionality of this enzyme. Substitutions of amino acid 97 (proline) appearing in the outer layer cause the appearance of a succession of mutations at positions 117 (histidine) and 128 (leucine), which extend into the enzyme. This is a very serious defect, as mutation changes to an amino acid belonging to the catalytic tetrads in this case result in the enzyme ceasing to perform its original function, which may lead to its inactivation. Table 4 shows the correlated mutations associated with amino acid changes at position 138 of the enzyme. If these mutations occur in 17-HSD type 1 (see Figure 5A,B), they would result in the deletion of leucine and valine ­ or they would be replaced by another amino acid. These mutations concern amino acids that lie in very close proximity just below the outer surface of the protein. The Figure 8:(A, B) Selected correlated mutations incorporated in Table 7 in the molecule of human 17-HSD type 1 (1ry0.pdb). Pink position ­ primary mutational change (glutamine-217); green position ­ correlated mutation with the primary mutation (alanine-230). consequence of this variability mutation may be a change in the spatial structure and in its functioning. The probability of a correlated mutation is also found in the deletion or change to an amino acid at position 153 of the protein (see Table 5). This mutations in human 17HSD type 1 (see Figure 6A,B) comprise amino acids on the surface, lying in close proximity; an aspartic acid mutation at position 153 would cause more mutations in this protein: leucine (149) and valine (154). The appearance of such correlated mutations into dehydrogenase 17HSD type 1 may result in changes in the spatial structure of the protein, distorting its functionality even though they do not cover the NADPH binding site or active site. If the correlated mutations in Table 6 occur in 17-HSD type 1 (see Figure 7A,B), they would result in the deletion yniewska et al.: Mutations in HSDs23 of valine and aspartic acid ­ or they would be replaced by another amino acid. The consequence of this variability mutation may be a change in the spatial structure and in its functioning. A mutation of the amino acid at position 217 (see Table 7) show the next possible location of the correlated mutation in the HSD family. If these mutations occur in 17-HSD type 1, they would be replaced by another amino acid ­ glutamine (position 217) and alanine (position 230) (Figure 8A,B). The appearance of such correlated mutations into dehydrogenase 17-HSD type 1 may result in changes in the spatial structure of the protein, distorting its functionality even though they do not cover the NADPH binding site or active site. Conclusions The correlated mutations in the primary structure of HSDs are located in distant sites. This makes the primary structure highly variable. This kind of study is the first approach for the HSD family. The identification and meticulous characterisation of variable mutation regions and conservative regions are valuable sources of information. All the collected and received information on this family of proteins may assist in the design and further modelling of new drugs. Author contributions: The authors accept responsibility for the entire content of this article and approved its submission. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organisation(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bio-Algorithms and Med-Systems de Gruyter

Correlated mutations in hydroxysteroid dehydrogenases family

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de Gruyter
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1895-9091
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10.1515/bams-2016-0024
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Abstract

Background: Hydroxysteroid dehydrogenase enzymes belong to the short-chain dehydrogenase/reductase (SDR) superfamily and aldo-keto reductases (AKRs). SDR is involved in the metabolism of many compounds (hormones, lipids, etc.) and is present in almost all studied genomes. Two hundred members of hydroxysteroid dehydrogenases have been analysed in terms of natural mutational variability. The second superfamily comprises AKR superfamily group enzymes whose function is catalysing the oxidation and reduction of many substrates by binding NAD(P)H as a cofactor. This kind of study is the first approach for the hydroxysteroid dehydrogenase family. This information grants practical meaning to designing potential specific drugs to fight specific diseases caused by mutations. Methods: In the research, amino acid sequences of representatives of the hydroxysteroid dehydrogenase family were extracted from the UniProt database. In total, the analysed 200 sequences with the highest degree of similarity were shown by BLAST searches. In the sequence analyses, we used the following software: ClustalX (multiple sequence alignment), Consensus Constructor (creating consensus sequence), and CORM (finding correlated mutations). Results: The CORM program identified potential sites of correlated mutations in hydroxysteroid dehydrogenases. This program generated 18 tables of results that contain the amino acid positions of mutations. Seven of these are presented in this paper. Conclusions: The primary structure of the hydroxysteroid dehydrogenase family shows high variation. Keywords: aldo-keto reductases; correlated mutations; hydroxysteroid dehydrogenases; short-chain dehydrogenase/reductase. *Corresponding author: Agata yniewska, Department of Molecular Biology, Faculty of Biological Sciences, University of Zielona Góra, Zielona Góra, Poland, E-mail: agaw0606@gmail.com Jacek Leluk and Gabriela aroffe: Department of Molecular Biology, Faculty of Biological Sciences, University of Zielona Góra, Zielona Góra, Poland Hydroxysteroid dehydrogenases HSDs have multiple functions. They play an important role in the local production of steroid hormones in target tissues; they can transform active sex hormones into inactive forms, and vice versa. They also have the function of catalysing stereoselective reactions at a specific site so that all steroid hormones possess the appropriate isoform that either inactivates hormones or activates the ligand form. HSDs have oxidative and reductive properties so that their isoforms can act as molecular switches for `on' and `off' [1, 2]. SDR superfamily SDR is one of the largest superfamilies of proteins. These enzymes were identified as a separate and new group of oxidoreductases at the end of the year 1979; the term SDR was coined in 1991 [1, 3]. One of the characteristics of these enzymes is that their chains reach a length of about 250 amino acids. HSDs, which belong to this family, have a single binding domain of NAD(P)(H) and exhibit a wide variety of substrates [4, 5]. Another significant feature of this protein superfamily is the presence of characteristic sequence motifs that are arranged in a certain way. These motifs include the highly conserved triad of Ser, Tyr, and Lys of the active site and the nucleotide-binding Rossmann motif. The Rossmann motif consists of six to seven chains, which are surrounded by three to five -helical chains. SDR enzymes are involved in the metabolism of many compounds (hormones, lipids, etc.) and are present in almost all studied genomes (in humans, they are coded by >70 genes). The SDR superfamily is divided into five classes: classic, intermediate, extensive, diverse, and complex. They catalyse the major steps of inactivation or 18yniewska et al.: Mutations in HSDs activation in vitamins, steroids, prostaglandins, and other bioactive molecules by reduction and oxidation, respectively, in carbonyl and hydroxyl groups [2, 6] (Table 1). result was subjected to manual verification. The result of the ClustalX program ­ the multiple sequence alignment ­ was used in the CORM program to identify potential sites of correlated mutations and was further analysed in Consensus Constructor. AKR superfamily This is a family of enzymes whose function is catalysing the oxidation and reduction of many substrates by binding NAD(P)H as a cofactor. AKRs are oxidoreductases that share a common protein structure, /-barrels, which consist of eight -helices and eight -chains that are arranged inside the structure for rigidity. The structure of /-barrel contains the active site and pocket binding of NAD(P)H. AKR family enzymes have three big loops in the C-ends of barrels. Changes in this region of the enzyme determine the affinity of the substrates [9­11] (Table 2). Computer programs used ­ ClustalX: This program was used to match homologous sequences and create multiple sequence alignments. CORM: This program was used to search, analyse, and describe correlated mutations occurring in families of homologous proteins. Consensus Constructor: This program was used to create a consensus sequence from a set of matched sequences as well as a detailed analysis of these matches. Chimera: This program was used to create figures with three-dimensional structures. Methods Databases and the leader sequence In the research, the amino acid sequences of members of the HSD family were extracted from the UniProt database available on the Internet [14]. In the next step, the downloaded sequences were tested using BLAST if they show affinity to known members of the dehydrogenases family [15, 16]. The analysis used 200 sequences with the highest degree of similarity. The HSD analysis group adopted the sequence leader, which was based on database searches. The leader sequence was: 3 -HSD/Delta 54-isomerase type 2 (identification no. P26439). Homologous sequences were arranged using the ClustalX program [17]. The multiple sequence alignment Table 1:Characteristics of the superfamily: SDR [7, 8]. Build Main structural motif Cofactor Binding site Multimeric (monomer 25 kDa) Rossmann motif (-)2 NAD(P)(H) Motif Tyr-X-X-X-Lys Results The CORM program identified potential sites of correlated mutations in HSDs. This program generated 18 tables of results. Seven of them are presented in this paper. Table 3 shows the number of possible consequences of amino acid changes at position 97 concerning cysteine (C) or tyrosine (Y). A change of an amino acid in this Table 3:First cluster of correlated mutations in the HSD family. Position Amino Counts 117: -ACEFHKMQRVWY 128: -FGILMVY acid 97 97 C Y 101 81 E ACFHKMQRWY LM FIVY Table 4:Second cluster of correlated mutations in the HSD family. Position 138 138 Amino acid ­ E Counts 79 73 137: -EGHIKLPQRSTV ­ EIKLQRV Table 2:Characteristics of the superfamily: AKR [10, 12, 13]. Build Main structural motif Cofactor Binding site Monomeric (34 kDa) TIM-barrel structure (/)8 NAD(P)(H) Conservative tetrad: Tyr55, Asp50, Lys84, His117 Table 5:Third cluster of correlated mutations in the HSD family. Position 153 153 Amino acid ­ T Counts 93 73 149: -KLQRV ­ KLQR 154: -AEKLMQ ­ AEKLMQ yniewska et al.: Mutations in HSDs19 Table 6:Fourth cluster of correlated mutations in the HSD family. Position 154 154 Amino acid ­ K Counts 94 89 153: -AIPSTV -P AISTV Table 8:Sixth cluster of correlated mutations in the HSD family. Position Amino Counts 308: -DEHKLNQRTVY 331: -CFLPQRSWY acid 294 294 G Y 73 EHKLNQRTVY 83 -D CFLRW -PQ Table 7:Fifth cluster of correlated mutations in the HSD family. Position Amino Counts 230: -ACFILMSTV 387: -CEIKLMNPQRSTV acid 217 217 G S 78 CILMSTV 67 A CEKMNPRST -ILQV position to cysteine may result in the appearance of a mutation coupled at position 117 with original amino acids: ACEFHKMQRVWY and deletion marked by -. Subsequently, in this place can occur glutamic acid (E), and at position 128: -FGILMVY, where leucine (L) or methionine (M) may occur. However, regarding the occurrence at position 97, the amino acid tyrosine (Y) may result in the occurrence of amino acid changes at position 117, -ACEFHKMQRVWY on ACFHKMQRWY, and the position 128: -FGILMVY on FIVY. Table 4 shows the correlated mutations associated with amino acid changes at position 138 of the enzyme. At this position may occur 138 amino acid deletions, the result of which will be the next amino acid deletion at position 137. If, however, this place is substituted with glutamic acid (138), mutations may occur in position 137, which will change the primary amino acid -EGHIKLPQRSTV to another: EIKLQRV. The probability of a correlated mutation is also found in the deletion or change to an amino acid at position 153 of the proteins (see Table 5). The deletion at this place leads to the deletion of the following positions: 149 -KLQRV and 154 -AEKLMQ. A change of 153 amino acid to threonine (T) causes a further change to the amino acid at position 149, -KLQRV on KLQR, and at position 154: -AEKLMQ on any amino acid from the same set AEKLMQ. A mutation to an amino acid at position 154 (see Table 6) may take place in two ways. In the first case, the deletion of amino acid 154 as a result of mutations may cause another change in position 153: -AIPSTV on the deletion of this amino acid or its change to proline (P). In the second instance, the 154 amino acid can lead to mutations in the adjacent position 153: -AIPSTV on AISTV. Table 7 shows the next possible location of the mutation correlated in the HSD family. A mutation of the amino acid at position 217 determines the subsequent emergence of mutations at positions 230 and 387 (see Figure 1). The next result of the CORM program is shown in Table 8: the mutation of position 294 can cause the following mutations (see Figure 2). The consequence of mutational changes in position 381 in the HSD family is the emergence of several correlated mutations (see Table 9). A change of amino acid 381 to serine (S) will trigger further changes in the following positions: ­ 239: -AFILMV will change amino acid on isoleucine (I), leucine (L), or valine (V). ­ 329: -ACHIQRSTV will change amino acid on alanine (A), isoleucine (I), or valine (V). Mutation at position 217 Change amino acid on glycine (G) Change amino acid on serine (S) Mutation at position 230:-ACFILMSTV Mutation at position 387:-CEIKLMNPQRSTV Amino acid deletion Change amino acid on CILMSTV Change amino acid on CEKMNPRST Change amino acid on A Change amino acid on ILQV Figure 1:Pathways of correlated mutations caused by amino acid mutation at position 217. 20yniewska et al.: Mutations in HSDs Change amino acid on glycine (G) Mutation at position 308:-DEHKLNQRTVY Mutation at position 331:-CFLPQRSWY Change amino acid on EHKLNQRTVY Change amino acid on CFLRW Mutation at position 294 Mutation at position 308:-DEHKLNQRTVY Change amino acid on tyrosine (Y) Mutation at position 331:-CFLPQRSWY Change amino acid on D Amino acid deletion Change amino acid on PQ Figure 2:Pathways of correlated mutations caused by amino acid mutation at position 294. Table 9:Seventh cluster of correlated mutations in the HSD family. Position 381 381 Amino acid S Y Counts 89 76 239: -AFILMV ILV FM 329: -ACHIQRSTV AIV HQRS 355: -AGMPS AG M 380: -ACILQSV ILV ACS 386: -ACFHILMPQSTVY HLPV CMSTY 427: -ACEFHILMNQTVY -ACLMQT HNY 461: -CFIMSTY -FMY ST ­ ­ ­ ­ ­ 355: -AGMPS will change amino acid on alanine (A) or glycine (G). 380: -ACILQSV will change amino acid on isoleucine (I), leucine (L), or valine (V). 386: -ACFHILMPQSTVY will change amino acid on histidine (H), leucine (L), proline (P), or valine (V). 427: -ACEFHILMNQTVY will change amino acid on A, C, L, M, Q, T, or the deletion. 461: -CFIMSTY will change amino acid on phenylalanine (F), methionine (M), tyrosine (Y), or the deletion. ­ ­ ­ ­ ­ ­ ­ Change of amino acid 381 to tyrosine (Y) will trigger further changes in positions, as follows: 239: -AFILMV will change amino acid on phenylalanine (F) or methionine (M). 329: -ACHIQRSTV will change amino acid on histidine (H), glutamine (Q), arginine (R), or serine (S). 355: -AGMPS will change amino acid on methionine (M). 380: -ACILQSV will change amino acid on alanine (A), cysteine, (C), or serine (S). 386: -ACFHILMPQSTVY will change amino acid on cysteine (C), methionine (M), serine (S), threonine (T), or tyrosine (Y). 427: -ACEFHILMNQTVY will change amino acid on histidine (H), asparagine (N), or tyrosine (Y). 461: -CFIMSTY will change amino acid on serine (S) or threonine (T). Figure 3:Consensus sequence. yniewska et al.: Mutations in HSDs21 Consensus sequence The analysed group of proteins created a list of about 200 sequences showing the closest similarity to the reference sequence. They were aligned by the ClustalX program. Further, a detailed verification of the matches was carried out manually. Then, they were used by the Consensus Constructor [18] program, which created a consensus sequence, a collective summary of the studied group of enzymes. The parameters for use were a minimum amino acid count of 66 and a maximum conservatism (%) of 80.0. In Figure 3 the black background shows a strong conservative place, the grey background shows a moderately conserved place, and the white background shows a high variability of the amino acid. Figure 4:Selected correlated mutations incorporated in Table 3 in the molecule of human 17-HSD type 1 (1ry0.pdb). Pink position ­ primary mutational change (proline-97); green position ­ correlated mutation with the primary mutation (histidine-117 and leucine-128). Figure 5:(A, B) Selected correlated mutations incorporated in Table 4 in the molecule of human 17-HSD type 1 (1ry0.pdb). Pink position ­ primary mutational change (leucine-138); green position ­ correlated mutation with the primary mutation (valine-137). Figure 6:(A, B) Selected correlated mutations incorporated in Table 5 in the molecule of human 17-HSD type 1 (1ry0.pdb). Pink position ­ primary mutational change (aspartic acid-153); green position ­ correlated mutation with the primary mutation (leucine-149 and valine-154). 22yniewska et al.: Mutations in HSDs Figure 7:(A, B) Selected correlated mutations incorporated in Table 6 in the molecule of human 17-HSD type 1 (1ry0.pdb). Pink position ­ primary mutational change (valine-154); green position ­ correlated mutation with the primary mutation (aspartic acid-153). Discussion In this study, we observed the correlated mutations in the HSD family. The occurrence of correlated mutations in 17-HSD type 1 in Table 3 (see Figure 4) may result in major changes in the functionality of this enzyme. Substitutions of amino acid 97 (proline) appearing in the outer layer cause the appearance of a succession of mutations at positions 117 (histidine) and 128 (leucine), which extend into the enzyme. This is a very serious defect, as mutation changes to an amino acid belonging to the catalytic tetrads in this case result in the enzyme ceasing to perform its original function, which may lead to its inactivation. Table 4 shows the correlated mutations associated with amino acid changes at position 138 of the enzyme. If these mutations occur in 17-HSD type 1 (see Figure 5A,B), they would result in the deletion of leucine and valine ­ or they would be replaced by another amino acid. These mutations concern amino acids that lie in very close proximity just below the outer surface of the protein. The Figure 8:(A, B) Selected correlated mutations incorporated in Table 7 in the molecule of human 17-HSD type 1 (1ry0.pdb). Pink position ­ primary mutational change (glutamine-217); green position ­ correlated mutation with the primary mutation (alanine-230). consequence of this variability mutation may be a change in the spatial structure and in its functioning. The probability of a correlated mutation is also found in the deletion or change to an amino acid at position 153 of the protein (see Table 5). This mutations in human 17HSD type 1 (see Figure 6A,B) comprise amino acids on the surface, lying in close proximity; an aspartic acid mutation at position 153 would cause more mutations in this protein: leucine (149) and valine (154). The appearance of such correlated mutations into dehydrogenase 17HSD type 1 may result in changes in the spatial structure of the protein, distorting its functionality even though they do not cover the NADPH binding site or active site. If the correlated mutations in Table 6 occur in 17-HSD type 1 (see Figure 7A,B), they would result in the deletion yniewska et al.: Mutations in HSDs23 of valine and aspartic acid ­ or they would be replaced by another amino acid. The consequence of this variability mutation may be a change in the spatial structure and in its functioning. A mutation of the amino acid at position 217 (see Table 7) show the next possible location of the correlated mutation in the HSD family. If these mutations occur in 17-HSD type 1, they would be replaced by another amino acid ­ glutamine (position 217) and alanine (position 230) (Figure 8A,B). The appearance of such correlated mutations into dehydrogenase 17-HSD type 1 may result in changes in the spatial structure of the protein, distorting its functionality even though they do not cover the NADPH binding site or active site. Conclusions The correlated mutations in the primary structure of HSDs are located in distant sites. This makes the primary structure highly variable. This kind of study is the first approach for the HSD family. The identification and meticulous characterisation of variable mutation regions and conservative regions are valuable sources of information. All the collected and received information on this family of proteins may assist in the design and further modelling of new drugs. Author contributions: The authors accept responsibility for the entire content of this article and approved its submission. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organisation(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

Journal

Bio-Algorithms and Med-Systemsde Gruyter

Published: Mar 1, 2017

References