* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download COMMUNICATION Engineering the Amine Transaminase from
Oligonucleotide synthesis wikipedia , lookup
Proteolysis wikipedia , lookup
Peptide synthesis wikipedia , lookup
Vesicular monoamine transporter wikipedia , lookup
Genetic code wikipedia , lookup
Biochemistry wikipedia , lookup
Ultrasensitivity wikipedia , lookup
Drug design wikipedia , lookup
Clinical neurochemistry wikipedia , lookup
Two-hybrid screening wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Catalytic triad wikipedia , lookup
Metalloprotein wikipedia , lookup
Enzyme inhibitor wikipedia , lookup
Point mutation wikipedia , lookup
Biosynthesis wikipedia , lookup
Ligand binding assay wikipedia , lookup
Discovery and development of neuraminidase inhibitors wikipedia , lookup
COMMUNICATION Engineering the Amine Transaminase from Vibrio fluvialis towards Branched-Chain Substrates Maika Genz,[a] Okke Melse,[a] Sandy Schmidt,[a] Clare Vickers,[a] Tom van den Bergh,[b] Henk-Jan Joosten,[b] and Uwe T. Bornscheuer*[a] Abstract: Chiral amines are important building blocks, especially for the pharmaceutical industry. Although amine transaminases (ATA) are versatile enzymes to synthesize chiral amines, the wild-type enzymes do not accept ketones with two large substituents next to the carbonyl function. Using bioinformatic tools to design a 7-site mutant library followed by high-throughput screening, we were able to identify variants with a widened binding pocket of the enzyme from Vibrio fluvialis (VF-ATA) as exemplified for a range of ketones. Three variants allow the asymmetric synthesis of tert-butylphenyl amine – not accessible by any wild-type ATA described so far. The best variant containing four mutations (L56V, W57C, F85V, V153A) gave 100% conversion of the ketone, yielding the amine with >99%ee, notably with a preference to the (R)-enantiomer. In silico modeling enabled the reconstruction of the substrate binding mode to the newly evolved binding pocket and hence to explain the experimental results. side chain present in amino acids such as alanine, commonly serving as amine donor, binding in the same pocket.[4] This dual substrate recognition is realized by a flexible arginine residue, called ‘flipping arginine’ (R415 in the enzyme from Vibrio fluvialis; VF-ATA). This arginine is located in the large binding pocket and either forms a salt bridge to the carboxylate group or moves out of the active site (Figure 1A). Chiral amines are versatile building blocks for the pharmaceutical and agrochemical industry. Hence, methods for their stereoselective synthesis are highly desired, especially to ensure that the desired biologically active enantiomer is solely produced. Beside the chemical asymmetric synthesis using stereoselective hydrogenation with transition-metal catalysts and enzymatic methods using e.g. imine reductases, amine dehydrogenases or monoamine oxidases, amine transaminases (ATA) have become an important alternative route for the synthesis of chiral amines.[1] The most popular example for the engineering of an ATA is the (R)-selective transaminase ATA117 developed by Merck & Co and Codexis, which contains 27 mutations to enable the biocatalytical production of the drug sitagliptin.[2] Notably, this enzyme belongs to fold class IV, whereas (S)-selective ATA belong to the structurally different fold class I. Transaminases are pyridoxal-5'-phosphate (PLP) dependent enzymes.[3] The active form of transaminases is known to be a homodimer with the active site at the dimer interface built up from residues of both monomers. The substrate binding site of ATAs is defined by a large and a small binding pocket (Figure 1). The small binding pocket provides space for either the alkyl side chain of the hydrophobic substrate or, alternatively, the acidic [a] [b] Dr. M. Genz, O. Melse, Dr. S. Schmidt, Dr. C. Vickers, Prof. Dr. U.T. Bornscheuer Department of Biotechnology & Enzyme Catalysis Institute of Biochemistry Felix-Hausdorff-Str. 4 D-17487 Greifswald (Germany) E-Mail: [email protected] http://biotech.uni-greifswald.de T. van den Bergh, Dr. H.-J. Joosten Bio-Prodict Nieuwe Marktstraat 54E 6511 AA Nijmegen (The Netherlands) Supporting information for this article is given via a link at the end of the document. Figure 1. Substrate binding pocket of the ATA from Vibrio fluvialis (VF-ATA; PDB-code: 4E3Q). A) Schematic view of the large and small binding pocket bearing the external aldimine intermediate (PLP-Schiff' base with (S)-1phenylethylamine). The catalytic lysine (K285) is highlighted in red. The targeted amino acids are colored (7-site library yellow, V153 green). The asterisk denotes residues, which are located in the second monomer forming the active site of the dimeric ATA. B) View of the substrate binding pocket. Important residues are shown as sticks: Pyridoxamine-5’-phosphate (PMP) in dark grey, 7-site library in yellow, V153 in green. When considering the benchmark substrate for transaminases, 1-phenylethylamine (1b, Scheme 1), the phenyl group is placed COMMUNICATION in the large binding pocket whereas the small binding pocket is mainly limited to a methyl group. In the past few years, a lot of effort has been undertaken to engineer transaminases of fold class I in such a way that the small binding pocket can accept bulky moieties like an ethyl (Scheme 1, 2b), propyl (3b) or ethanol side chain, albeit with limited success, especially if larger substituents are present.[4b, 5] Recently, we have succeeded in engineering the (S)-selective ATA from Ruegeria sp. (pdb-code 3FCR) to enable the synthesis of a range of bulky amines with high to excellent enantioselectivity. [6] For this purpose only five mutations were required and to achieve reasonable activity only two mutations (Y59W/T231A in 3FCR, corresponding to W57/A228 in VF-ATA) were sufficient. Interestingly, these two residues are present in wild-type VFATA, but this enzyme has no activity towards bulky substrates. As VF-ATA is one of the most frequently used (S)-ATAs, this lack of activity prompted us to subject this enzyme to extensive protein engineering to make it capable to accept the bulky substrates 3b–6b (Scheme 1) beyond the compounds mentioned above – including 2b. Thus far the conversion of branched chained substrates could not be achieved.[7] The only exception is a variant of ATA-117, which gave 56% conversion for phenylisobutylamine (4b).[2] NH2 1b NH2 4b NH2 2b NH2 5b NH2 3b NH2 6b Scheme 1. Target amines for the iterative extension of the binding pocket of the VF-ATA. The box highlights the motif, which binds in the small pocket. To achieve this goal, our protein design strategy consisted of three steps: (i) enlarging the volume of the substrate binding pocket by creation of a library including mainly hydrophobic residues in the active site, (ii) optimization of the hits for asymmetric synthesis and (iii) combination of all findings to create a synthetically useful final variant. In order to identify most suitable mutation sites and amino acid substitutions in the ATA from Vibrio fluvialis, we have performed a bioinformatic analysis using 3DM. 3DM is a protein superfamily analysis suite, which uses highly sophisticated algorithms for the calculation of alignments based on structure/sequence-function relationships of a defined superfamily.[8] A database on the PLP-dependent transferases of fold type I was used, which contains 725 structures to which 61,560 sequences were aligned.[3] Within this superfamily database, a sub-dataset based on the VF-ATA (pdb-code: 4E3Q[5a]) was created with 12,956 aligned sequences. This inspection identified seven positions (F19, L56, W57, F85, F86, R415, and L417) within the binding pocket of VF-ATA – all containing important hydrophobic residues involved in substrate binding in the active site (Figure 1). These positions were then subjected to mutagenesis using distinct degenerate codons encoding hydrophobic amino acids selected based on the 3DM analysis (Supporting Information, Table S9). Thereby the active site is widened by the introduction of smaller hydrophobic amino acids, while preserving the hydrophobic network, which is important for the interaction of both monomers creating the active site. After expression in E. coli, this 7-site library comprising 16,384 mutations/combinations was screened at our fully automated robotic platform (http://lara.uni-greifswald.de) using rac-3b or rac-4b as amine donors in the kinetic resolution mode.[9] Because the flipping arginine (R415) was included in the library, screening was performed not only with pyruvate, but also with pentanal as amine acceptor as this is only accepted if the R415 is mutated.[5d] The screening of 2,240 variants (4.5% coverage of the total library) already resulted in the identification of five hits, which contained up to five mutations in the positions L56, W57, F85, R415 and L417 (Supporting Information, Tables S1 and S2). From these hits the variants H1 (L56V, W57F, F85V, R415C) and H3 (L56V, W57C, R415F, L417A) were the most active ones as both of them showed an increase in activity using both amines 3b and 4b (Table 1 and Supporting Information, Tables S3 and S4). Calculation of the binding pocket volume indeed revealed the desired increase: the active site of H1 was enlarged 1.4-fold (122 Å3) and for H3 3.1-fold (275 Å3) compared to the wild type (88 Å3) (Supporting Information, Table S5).[10] In the second screening round, conversion of the more sterically demanding amines 5b and 6b was targeted. It was known that the change of V153 to alanine is beneficial for the conversion of bulky substrates.[5a,7] Therefore, the mutations V153A and – to create even more space – V153G were introduced into H1 and H3 resulting in H1_A or H1_G and H3_A or H3_G, respectively (see Table S1 in the Supporting Information for the nomenclature and mutations present in all variants). Comparing the variants containing V153A or V153G, it becomes obvious that the V153A mutation is beneficial (H1_A shows a 12-fold increase in activity towards amine 4b compared to H1; H3_A of about 2-fold) whereas the V153G mutation only gave a 2-fold improvement over H1 and even a decrease in activity using H3_G compared to H3 (Supporting Information, Table S4). This can be attributed to the possible role of V153 in dimer formation, which can also explain the loss in protein stability for V153G (Supporting Information, Table S8 and Figure S3). At this point it was noticed that the change of the flipping arginine R415 to either phenylalanine or cysteine lead to a loss of activity in asymmetric synthesis using alanine as amine donor (see Supporting Information). Thus, residue R415 was backmutated in variant H1 leading to H1_R and in H3 (including the back-mutation of L417) leading to H3_R. We already showed that position L417 does not have a positive effect when widening the substrate binding pocket.[5d] COMMUNICATION Table 1. Specific activity (As) of the most important variants determined in kinetic resolution. Variants were tested with the corresponding amine using either pyruvate or pentanal as amine acceptor. For determination of the activity all variants were produced in E. coli and purified.[5d] Enzyme As[a] [U mg-1] As [U mg-1] As [U mg-1] As [U mg-1] As [U mg-1] As [U mg-1] variant pyruvate : 4b pyruvate : 5b pyruvate : 6b pentanal : 4b pentanal : 5b pentanal : 6b [b] Wt 0.056 ± 0.008 n.a. n.a. 0.045 ± 0.010 n.a. n.a. H1 0.104 ± 0.005 0.008 ± 0.002 n.a. 0.140 ± 0.011 0.054 ± 0.002 n.a. H3 0.055 ± 0.009 n.a. n.a. 0.118 ± 0.013 n.a. n.a. H1_A 0.064 ± 0.007 0.006 ± 0.001 n.a. 1.642 ± 0.048 0.055 ± 0.005 0.302 ± 0.013 H1_R 0.009 ± 0.005 0.013 ± 0.001 0.012 ± 0.002 0.063 ± 0.031 0.046 ± 0.003 n.a. H3_A 0.077 ± 0.002 n.a. 0.009 ± 0.004 0.232 ± 0.028 0.030 ± 0.004 0.044 ± 0.020 H3_R 0.072 ± 0.007 0.012 ± 0.001 n.a. 0.077 ± 0.013 n.a. n.a. H3_RV 0.047 ± 0.007 0.0336 ± 0.0001 n.a. 0.081 ± 0.022 0.025 ± 0.001 n.a. H3_RA 0.121 ± 0.015 0.0780 ± 0.0003 n.a. 0.127 ± 0.016 0.0235 ± 0.0009 n.a. H3_RAV 0.181 ± 0.016 0.118 ± 0.003 0.036 ± 0.015 0.288 ± 0.067 0.067 ± 0.001 n.a. [a] As = Specific activity where 1 U corresponds to 1 µmol product formation per min; [b] n.a.: not active / below detection limit; more detailed information about all variants can be found in the supporting information in Table S3 (with pyruvate) and Table S4 (with pentanal) as amine acceptor. Reconstruction of R415 in both variants does not have a positive influence in kinetic resolution mode (Table 1), but in asymmetric synthesis (Table 2). Comparison of all variants for amine 5b revealed H1_R and H3_R as best variants (Table 1, pyruvate:5b). Thus, we focused on the variants containing the back-mutated R415 as only these can be used for asymmetric synthesis with alanine. Calculation of the binding pocket volume of the variants H1_R and H3_R resulted in 113 A³ and 175 A³, respectively (Supporting Information, Table S5).[10] This finding reveals that the W57C mutation in H3_R creates even more space in the binding pocket compared to both mutations of H1_R, W57F and F85V (L56V is present in both variants). The specific activity for both variants concerning amine 5b and pyruvate is almost equal, concerning 6b only H1_R shows some minor activity (Table 1, pyruvate as acceptor). Finally, the best two variants were combined for the creation of a larger binding pocket: H3_R was used as template, as it has a larger binding pocket than H1_R and can perform asymmetric synthesis in contrast to H3. H3_R was complemented with either the beneficial mutation of V153A (resulting in H3_RA) or the hotspot from the variant H1, which is F85V (resulting in H3_RV) or both (resulting in H3_RAV). All of these new variants showed activity using amines 4b and 5b – with highest activity for the H3_RAV variant (Table 1, pyruvate; 181 mU mg-1 for 4b and 118 mU for 5b). For the most interesting amine 6b, only the H3_RAV variant showed activity (Table 1, pyruvate; 36 mU mg-1). Interestingly, variant H1_R also showed activity with amine 6b (Table 1, pyruvate; 12 mU mg-1). Variants H2, H3_A and H3_G also showed some minor activity using 6b in kinetic resolution, but as they still contained mutations at position R415, they were not considered for asymmetric synthesis. With these results achieved, we performed asymmetric synthesis of 4b–6b using only the variants having the R415 intact (Table 2). From all variants, H1_R, H3_RA and H3_RAV showed conversion for the targeted amines. Wild-type VF-ATA gave only 6% conversion after 8d using 4a, and no conversion at all for 5a and 6a. Again, for all three amines the H3_RAV variant was the best enzyme resulting in 74% conversion for 4b, 87% conversion for 5b and full conversion of 6b, (for 6b notably accomplished after only 48 h, Supporting Information Table S6). Here, all active mutants gave the (R)-enantiomer with excellent (99% ee) optical purity (Table 2). In case of 4b and 5b optical purities were lower, but as our protein engineering strategy targeted to find active enzyme variants, it is not surprising that they might not exhibit also high enantioselectivity. A closer look to the substrate binding revealed that 6b is more restrained to the active conformation and thus fits better in the binding pocket compared to amine 4b and 5b (Supporting Information, Table S7). Table 2. Asymmetric synthesis using wild-type ATA and the lead variants. Substrate Variant 4b 5b 6b Conv.[a] Optical purity[b] Conv. Optical purity Conv. Optical purity [%] [%ee] [%] [%ee] [%] [%ee] – n.a. – [c] [d] Wt 6 n.d. n.a. H1_R 18 19 39 n.d. 77 > 99 (R) H3_R 10 n.d. n.a. – n.a. – H3_RV 4 n.d. 15 n.d. n.a. – H3_RA 42 80 11 n.d. 25 > 99 (R) H3_RAV 74 50[e] 87 > 13 100 > 99 (R) [a] Conv. = conversion after 8 d (determined by GC) [b] ee = enantiomeric excess, [c] n.d. = not determined [d] n.a. = not active or below detection limit, [e] switch in enantiopreference compared to H1_R and H3_RV. Reaction conditions and further details for asymmetric synthesis are given in the Supporting Information (Table S6). It should be noted, that H1 and H3 were not active in asymmetric synthesis, due to the mutation of R415(C/F). COMMUNICATION [6] In summary, a combination of a ‘small, but smart’ library design by 3DM-based bioinformatic analysis with in silico studies resulted in the creation of variants of the amine transaminase from Vibrio fluvialis, able to catalyze the asymmetric synthesis of sterically demanding branched-chain chiral amines. Although only activity was a design target, we were very pleased to find that in case of tert-butylphenylamine also excellent optical purity could be achieved. Experimental Section All experimental details are provided in the Supporting Information. Acknowledgements We thank the European Union (KBBE-2011-5, Grant No. 289350), the DFG (INST 292/118-1 FUGG) and the federal state Mecklenburg-Vorpommern for financial support. We gratefully acknowledge Byung-Gee Kim (Seoul National University, Seoul, Korea) for providing the gene encoding the ATA from Vibrio fluvialis, Dr. Hans Iding (Hoffman LaRoche Ltd., Basel, Switzerland) for providing the pure enantiomer of (S)-6b, Dr. Ingrid Costa for assistance during synthesis of amine 5b and Ina Menyes for help during GC analysis. Additionally, we thank Prof. Dr. Matthias Höhne (Greifswald University, Greifswald, Germany) and Dr. Ioannis Pavlidis (Kassel University, Kassel, Germany) for fruitful discussions. Keywords: bioinformatics • chiral amines • enzyme catalysis • protein engineering • transaminase [1] [2] [3] [4] [5] a) R. C. Simon, N. Richter, E. Busto, W. Kroutil, ACS Catal. 2014, 4, 129–143; b) S. Mathew, H. Yun, ACS Catal. 2012, 2, 993–1001; c) H. Kohls, F. Steffen-Munsberg, M. Höhne, Curr. Opin. Chem. Biol. 2014, 19, 180–192; d) M. Fuchs, J. E. Farnberger, W. Kroutil, European J. Org. Chem. 2015, 6965–6982; e) A. A. Desai, Angew. Chem. Int. Ed. 2011, 50, 1974-1976; Angew. Chem. 2011, 123, 2018–2020; f) D. Wetzl, M. Gand, A. Ross, H. Müller, P. Matzel, S. P. Hanlon, M. Müller, B. Wirz, M. Höhne. H. Iding, ChemCatChem 2016, 8, 2023–2026; g) M. J. Abrahamson, E. Vázquez-Figueroa, N.B. Woodall, J.C. Moore, A.S. Bommarius, Angew. Chem. Int. Ed. 2012, 51, 3969–3972; Angew. Chem. 2012, 124, 4036–4040. C. K. Savile, J. M. Janey, E. C. Mundorff, J. C. Moore, S. Tam, W. R. Jarvis, J. C. Colbeck, A. Krebber, F. J. Fleitz, J. Brands, et al., Science 2010, 329, 305–309. F. Steffen-Munsberg, C. Vickers, H. Kohls, H. Land, H. Mallin, A. Nobili, L. Skalden, T. van den Bergh, H.-J. Joosten, P. Berglund, et al., Biotechnol. Adv. 2015, 33, 566–604. a) F. Steffen-Munsberg, C. Vickers, A. Thontowi, S. Schätzle, T. Meinhardt, M. Svedendahl Humble, H. Land, P. Berglund, U. T. Bornscheuer, M. Höhne, ChemCatChem 2013, 5, 154–157; b) J. S. Shin, B. G. Kim, J. Org. Chem. 2002, 67, 2848–2853. a) K. S. Midelfort, R. Kumar, S. Han, M. J. Karmilowicz, K. McConnell, D. K. Gehlhaar, A. Mistry, J. S. Chang, M. Anderson, A. Villalobos, et al., Protein Eng. Des. Sel. 2013, 26, 25–33; b) A. Nobili, F. SteffenMunsberg, H. Kohls, I. Trentin, C. Schulzke, M. Höhne, U. T. Bornscheuer, ChemCatChem 2015, 7, 757–760; c) S.-W. Han, E.-S. Park, J.-Y. Dong, J.-S. Shin, Adv. Synth. Catal. 2015, 357, 2712–2720; d) M. Genz, C. Vickers, T. van den Bergh, H.-J. Joosten, M. Dörr, M. Höhne, U. Bornscheuer, Int. J. Mol. Sci. 2015, 16, 26953–26963; e) S. Chakraborty, D. S. Touw, A. F. Peacock, J. Stuckey, V. L. Pecoraro, J. Am. Chem. Soc. 2010, 132, 13240–13250. [7] [8] [9] [10] I. V. Pavlidis, M. S. Weiß, M. Genz, P. Spurr, S. P. Hanlon, B. Wirz, H. Iding, U. T. Bornscheuer, Nat. Chem. 2016, DOI: 10.1038/NCHEM.2578. E. S. Park, S. R. Park, S. W. Han, J. Y. Dong, J. S. Shin, Adv. Synth. Catal. 2014, 356, 212–220. R. K. Kuipers, H. J. Joosten, W. J. H. Van Berkel, N. G. H. Leferink, E. Rooijen, E. Ittmann, F. Van Zimmeren, H. Jochens, U. Bornscheuer, G. Vriend, et al., Proteins Struct. Funct. Bioinforma. 2010, 78, 2101–2113. a) M. Dörr, M. P. C. Fibinger, D. Last, S. Schmidt, J. Santos-Aberturas, D. Böttcher, A. Hummel, C. Vickers, M. Voss, U. T. Bornscheuer, Biotechnol. Bioeng. 2016, 113, 1421–1432; b) S. Schätzle, M. Höhne, E. Redestad, K. Robins, U. T. Bornscheuer, Anal. Chem. 2009, 81, 8244–8248. J. D. Durrant, C. A. F. De Oliveira, J. A. McCammon, J. Mol. Graph. Model. 2011, 29, 773–776. COMMUNICATION Entry for the Table of Contents COMMUNICATION Based on bioinformatic analysis of the binding site of amine transaminases, a 7-site combinatorial library of the enzyme from V. fluvialis was created. High-throughput screening of this library led to the identification of enzyme variants able to convert sterically demanding ketones to afford the corresponding chiral amines in high to excellent optical purities and yields. M. Genz, O. Melse, S. Schmidt, C. Vickers, T. van den Bergh, H.-J. Joosten, U.T. Bornscheuer* Page No. – Page No. Engineering the Amine Transaminase from Vibrio fluvialis towards Branched Chain Substrates