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Significant improvement of the enantioselectivity of a halohydrin dehalogenase for asymmetric epoxide ring opening reactions by protein engineering

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Abstract

Halohydrin dehalogenases (HHDHs) have attracted much attention due to their ability to synthesize enantiomerically enriched epoxides and β-haloalcohols. However, most of the HHDHs exhibit low enantioselectivity. Here, a HHDH from the alphaproteobacteria isolate 46_93_T64 (AbHHDH), which shows only poor enantioselectivity in the catalytic resolution of rac-PGE (E = 9.9), has been subjected to protein engineering to enhance its enantioselectivity. Eight mutants (R89K, R89Y, V137I, P178A, N179Q, N179L, F187L, F187A) showed better enantioselectivity than the wild type. The best single mutant N179L (E = 93.0) showed a remarkable 9.4-fold increase in the enantioselectivity. Then, the single mutations were combined to produce the double, triple, quadruple, and quintuple mutants. Among the combinational mutants, the best variant (R89Y/N179L) showed an increased E value of up to 48. The E values of the variants N179L and R89Y/N179L for other epoxides 2–7 were 12.2 to > 200, which showed great improvement compared to 1.2 to 10.5 for the wild type. Using the variant N179L, enantiopure (R)-PGE with > 99% ee could be readily prepared, affording a high yield and a high concentration.

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References

  1. Chen CS, Fujimoto Y, Girdaukas G, Sih CJ (1982) Quantitative analyses of biochemical kinetic resolutions of enantiomers. J Am Chem Soc 104:7294–7299. https://doi.org/10.1021/ja00389a064

  2. de Jong RM, Tiesinga JJW, Rozeboom HJ, Kalk KH, Tang LX, Janssen DB, Dijkstra BW (2003) Structure and mechanism of a bacterial haloalcohol dehalogenase: a new variation of the short-chain dehydrogenase/reductase fold without an NAD(P)H binding site. EMBO J 22:4933–4944. https://doi.org/10.1093/emboj/cdg479

  3. de Jong RM, Tiesinga JJW, Villa A, Tang LX, Janssen DB, Dijkstra BW (2005) Structural basis for the enantioselectivity of an epoxide ring opening reaction catalyzed by haloalcohol dehalogenase HheC. J Am Chem Soc 127:13338–13343. https://doi.org/10.1021/ja0531733

  4. de Morais JWG, Maia AM, Martins PA, Fernandez-Lorente G, Guisan JM, Pessela BC (2018) Influence of different immobilization techniques to improve the enantioselectivity of lipase from Geotrichum candidum applied on the resolution of mandelic acid. Mol Catal 458:89–96. https://doi.org/10.1016/j.mcat.2018.07.024

  5. Gao PF, Li AT, Lee H, Wang DIC, Li Z (2014) Enhancing enantioselectivity and productivity of P450-catalyzed asymmetric sulfoxidation with an aqueous/ionic liquid biphasic system. ACS Catal 4:3763–3771. https://doi.org/10.1021/cs5010344

  6. Godinho LF, Reis CR, Rozeboom HJ, Dekker FJ, Dijkstra BW, Poelarends GJ, Quax WJ (2012) Enhancement of the enantioselectivity of carboxylesterase a by structure-based mutagenesis. J Biotechnol 158:36–43. https://doi.org/10.1016/j.jbiotec.2011.12.026

  7. Gu JL, Ye LD, Guo F, Lv XM, Lu WQ, Yu HW (2015) Rational design of esterase BioH with enhanced enantioselectivity towards methyl (S)-o-chloromandelate. Appl Microbiol Biotechnol 99:1709–1718. https://doi.org/10.1007/s00253-014-5995-x

  8. Guo F, Xu HM, Xu HN, Yu HW (2013) Compensation of the enantioselectivity-activity trade-off in the directed evolution of an esterase from Rhodobacter sphaeroides by site-directed saturation mutagenesis. Appl Microbiol Biotechnol 97:3355–3362. https://doi.org/10.1007/s00253-012-4516-z

  9. Guo F, Franzen S, Ye LD, Gu JL, Yu HW (2014) Controlling enantioselectivity of esterase in asymmetric hydrolysis of aryl prochiral diesters by introducing aromatic interactions. Biotechnol Bioeng 111:1729–1739. https://doi.org/10.1002/bit.25249

  10. Guo C, Chen YP, Zheng Y, Zhang W, Tao YW, Feng J, Tang LX (2015) Exploring the enantioselective mechanism of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by iterative saturation mutagenesis. Appl Environ Microb 81:2919–2926. https://doi.org/10.1128/aem.04153-14

  11. Hasnaoui-Dijoux G, Elenkov MM, Lutje Spelberg JH, Hauer B, Janssen DB (2008) Catalytic promiscuity of halohydrin dehalogenase and its application in enantioselective epoxide ring opening. ChemBioChem 9:1048–1051. https://doi.org/10.1002/cbic.200890021

  12. Hopmann KH, Himo F (2008a) Quantum chemical modeling of the dehalogenation reaction of haloalcohol dehalogenase. J Chem Theory Comput 4:1129–1137. https://doi.org/10.1021/ct8000443

  13. Hopmann KH, Himo F (2008b) Cyanolysis and azidolysis of epoxides by haloalcohol dehalogenase: theoretical study of the reaction mechanism and origins of regioselectivity. Biochemistry 47:4973–4982. https://doi.org/10.1021/bi800001r

  14. Hu D, Tang CD, Li C, Kan TT, Shi XL, Feng L, Wu MC (2017) Stereoselective hydrolysis of epoxides by reVrEH3, a novel Vigana radiate epoxide hydrolase with high enantioselectivity or high and complementary regioselectivity. J Agric Food Chem 65:9861–9867. https://doi.org/10.1021/acs.jafc.7b03804

  15. Kong XD, Zhou JH, Zeng BB, Xu JH (2014) A smart library of epoxide hydrolase variants and the top hits for synthesis of (S)-β-blocker precursors. Angew Chem Int Ed 53:6641–6644. https://doi.org/10.1002/ange.201402653

  16. Koopmeiners J, Halmschlag B, Schallmey M, Schallmey A (2016) Biochemical and biocatalytic characterization of 17 novel halohydrin dehalogenases. Appl Microbiol Biotechnol 100:7517–7527. https://doi.org/10.1007/s00253-016-7493-9

  17. Koopmeiners J, Diederich C, Solarczek J, Voβ H, Mayer J, Blankenfeldt W, Schallmey A (2017) HheG, a halohydrin dehalogenase with activity on cyclic epoxides. ACS Catal 7:6877–6886. https://doi.org/10.1021/acscatal.7b01854

  18. Li FH, Kong XD, Chen Q, Zheng YC, Xu Q, Chen FF, Fan LQ, Lin GQ, Zhou JH, Yu HL, Xu JH (2018) Regioselectivity engineering of epoxide hydrolase:near-perfect enantioconvergence through a single site mutation. ACS Catal 8:8314–8317. https://doi.org/10.1021/acscatal.8b02622

  19. Liu ZQ, Wu L, Zhang XJ, Xue YP, Zheng YG (2017) Directed evolution of carbonyl reductase from Rhodosporidium toruloides and its application in stereoselective synthesis of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate. J Agric Food Chem 65:3721–3729. https://doi.org/10.1021/acs.jafc.7b00866

  20. Ma HR, Yang X, Lu Z, Liu N, Chen YJ (2014) The “gate keeper” role of Trp222 determines the enantiopreference of diketoreductase toward 2-chloro-1-phenylethanone. PLoS One 9:e103792. https://doi.org/10.1371/journal.pone.0103792

  21. Mikleusevic A, Primozic I, Hrenar T, Salopek-Sondi B, Tang LX, Elenkov MM (2016) Azidolysis of epoxides catalyzed by the halohydrin dehalogenase from Arthrobacter sp. AD2 and a mutant with enhanced enantioselectivity: an (S)-selective HHDH. Tetrahedron:Asymmetr 27:930–935. https://doi.org/10.1016/j.tetasy.2016.08.003

  22. Nobili A, Gall MG, Pavlidis LV, Thompson ML, Schmidt M, Bornscheuer UT (2013) Use of ‘small but smart’ libraries to enhance the enantioselectivity of an esterase from Bacillus stearothermophilus towards tetrahydrofuran-3-yl-acetate. FEBS J 280:3084–3093. https://doi.org/10.1111/febs.12137

  23. Patel RN (2011) Biocatalysis: synthesis of key intermediates for development of pharmaceuticals. ACS Catal 1:1056–1074. https://doi.org/10.1021/cs200219b

  24. Pavlidis IV, Weiβ MS, Genz M, Spurr P, Hanlon SP, Wirz B, Iding H, Bornscheuer UT (2016) Identification of (S)-selective transaminases for the asymmetric synthesis of bulky chiral amines. Nat Chem 8:1076–1082. https://doi.org/10.1038/nchem.2578

  25. Reetz MT, Bocola M, Wang LW, Sanchis J, Cronin A, Arand M, Zou JY, Archelas A, Bottalla AL, Naworyta A, Mowbray S (2009) Directed evolution of an enantioselective epoxide hydrolase: uncovering the source of enantioselectivity at each evolutionary stage. J Am Chem Soc 131:7334–7343. https://doi.org/10.1021/ja809673d

  26. Savile CK, Janey JM, Mundorff EC, Moore JC, Tam S, Jarvis WR, Colbeck JC, Krebber A, Fleitz FJ, Brands J, Devine PN, Huisman GW, Hughes GJ (2010) Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science 329:305–309. https://doi.org/10.1126/science.1188934

  27. Schallmey A, Schallmey M (2016) Recent advances on halohydrin dehalogenases-from enzyme identification to novel biocatalytic applications. Appl Microbiol Biotechnol 100:7827–7839. https://doi.org/10.1007/s00253-016-7750-y

  28. Schallmey M, Koopmeiners J, Wells E, Wardenga R, Schallmey A (2014) Expanding the halohydrin dehalogenase enzyme family: identification of novel enzymes by database mining. Appl Environ Microbiol 80:7303–7315. https://doi.org/10.1128/AEM.01985-14

  29. Spickermann D, Hausmann S, Degering C, Schwaneberg U, Leggewie C (2014) Engineering of highly selective variants of Parvibaculum lavamentivorans alcohol dehydrogenase. ChemBioChem 15:2050–2052. https://doi.org/10.1002/cbic.201402216

  30. Sun HH, Zhang HF, Ang EH, Zhao HM (2018) Biocatalysis for the synthesis of pharmaceuticals and pharmaceutical intermediates. Bioorgan Med Chem 26:1275–1284. https://doi.org/10.1016/j.bmc.2017.06.043

  31. Tang LX, Zhu XC, Zheng HR, Jiang RX, Elenkov MM (2012) Key residues for controlling enantioselectivity of halohydrin dehalogenase from Arthrobacter sp. AD2 revealed by structure-guided directed evolution. Appl Environ Microb 78:2631–2637. https://doi.org/10.1128/AEM.06586-11

  32. Tentori F, Brenna E, Colombo D, Crotti M, Gatti FG, Ghezzi MC, Pedrocchi-Fantoni G (2018) Biocatalytic approach to chiral β-nitroalcohols by enantioselective alcohol dehydrogenase-mediated reduction of α-nitroketones. Catalysts 8:308–318. https://doi.org/10.3390/catal8080308

  33. Ulrich A, Volker M, Manfred PS (1993) Chromatographic resolution of chiral intermediates in β-adrenergic blocker synthesis on chiral stationary phases. Chirality 5:554–559. https://doi.org/10.1002/chir.530050712

  34. van Hylckama Vlieg JET, Tang LX, Lutje Spelberg JH, Smilda T, Poelarends GJ, Bosma T, Merode AEJ, Fraaije MW, Janssen DB (2001) Halohydrin dehalogenases are structurally and mechanistically related to short-chain dehydrogenases/reductases. J Bacteriol 183:5058–5066. https://doi.org/10.1128/JB.183.17.5058-5066.2001

  35. van Loo B, Lutje Spelberg JH, Kingma J, Sonke T, Wubbolts MG, Janssen DB (2004) Directed evolution of epoxide hydrolase from A. radiobacter toward higher enantioselectivity by error-prone PCR and DNA shuffling. Chem Biol 11:981–990. https://doi.org/10.1016/j.chembiol.2004.04.019

  36. Wan NW, Liu ZQ, Xue F, Huang K, Tang LJ, Zheng YG (2015) An efficient high-throughput screening assay for rapid directed evolution of halohydrin dehalogenase for preparation of β-substituted alcohols. Appl Microbiol Biotechnol 99:4019–4029. https://doi.org/10.1007/s00253-015-6527-z

  37. Watanabe F, Yu F, Ohtaki A, Yamanaka Y, Noguchi K, Odaka M, Yohda M (2016) Improvement of enantioselectivity of the B-type halohydrin hydrogen-halidelyase from Corynebacterium sp. N-1074. J Biosci Bioeng 122:270–275. https://doi.org/10.1016/j.jbiosc.2016.02.003

  38. Wu SK, Li AT, Chin YS, Li Z (2013) Enantioselctive hydrolysis of racemic and meso-epoxides with recombinant Escherichia coli expressing epoxide hydrolase from Sphinggomonas sp. HXN-200: preparation of epoxides and vicinal diols in high ee and high concentration. ACS Catal 3:752–759. https://doi.org/10.1021/cs300804v

  39. Wu ZY, Deng WF, Tong YP, Liao Q, Xin DM, Yu HS, Feng J, Tang LX (2017) Exploring the thermostable properties of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by a combinatorial directed evolution strategy. Appl Microbiol Biotechnol 101:3201–3211. https://doi.org/10.1007/s00253-017-8090-2

  40. Xue YP, Shi CC, Xu Z, Jiao B, Liu ZQ, Huang JF, Zheng YG, Shen YC (2015) Design of nitrilases with superior activity and enantioselectivity towards sterically hindered nitrile by protein engineering. Adv Synth Catal 357:1741–1750. https://doi.org/10.1002/adsc.201500039

  41. Xue F, Gao J, Zhang L, Li H, Huang H (2018a) Identification and characterization of a novel halohydrin dehalogenase from Bradyrhizobium erythrophlei and its performance in preparation of both enantiomers of epichlorohydrin. Catal Lett 148:1181–1189. https://doi.org/10.1007/s10562-017-2292-1

  42. Xue F, Ya XJ, Tong Q, Xiu YS, Huang H (2018b) Heterologous overexpression of Pseudomonas umsongensis halohydrin dehalogenase in Escherichia coli and its application in epoxide asymmetric ring opening reactions. Process Biochem 75:139–145. https://doi.org/10.1016/j.procbio.2018.09.018

  43. Xue F, Ya XJ, Xiu YS, Tong Q, Wang YQ, Zhu XH, Huang H (2019) Exploring the biocatalytic scope of a novel enantioselective halohydrin dehalogenase from an alphaproteobacterium. Catal Lett 149:629–637. https://doi.org/10.1007/s10562-019-02659-0

  44. You ZY, Liu ZQ, Zheng YG (2013) Properties and biotechnological applications of halohydrin dehalogenases: current state and future perspectives. Appl Microbiol Biotechnol 97:9–21. https://doi.org/10.1007/s00253-012-4523-0

  45. Zhang XJ, Shi PX, Deng HZ, Wang XX, Liu ZQ, Zheng YG (2018) Biosynthesis of chiral epichlorohydrin using an immobilized halohydrin dehalogenase in aqueous and non-aqueous phase. Bioresour Technol 263:483–490. https://doi.org/10.1016/j.biortech.2018.05.027

  46. Zhang XJ, Deng HZ, Liu N, Gong YC, Liu ZQ, Zheng YG (2019) Molecular modification of a halohydrin dehalogenase for kinetic regulation to synthesize optically pure (S)-epichlorohydrin. Bioresour Technol 276:154–160. https://doi.org/10.1016/j.biortech.2018.12.103

  47. Zhao J, Chu YY, Li AT, Ju X, Kong XD, Pan J, Tang Y, Xu JH (2011) An unusual (R)-selective epoxide hydrolase with high activity for facile preparation of enantiopure glycidyl ethers. Adv Synth Catal 353:1510–1518. https://doi.org/10.1002/adsc.201100031

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Xue, F., Zhang, L. & Xu, Q. Significant improvement of the enantioselectivity of a halohydrin dehalogenase for asymmetric epoxide ring opening reactions by protein engineering. Appl Microbiol Biotechnol (2020) doi:10.1007/s00253-020-10356-x

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Keywords

  • Halohydrin dehalogenase
  • Enantioselectivity
  • Saturation mutagenesis
  • Phenyl glycidyl ethers
  • Kinetic resolution