Laboratory evolution of an alcohol dehydrogenase towards enantioselective reduction of difficult-to-reduce ketones

Abstract

Background

A thermostable alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) has been repurposed to perform asymmetric reduction of a series of prochiral ketones with the formation of enantio-pure secondary alcohols, which are crucial chiral synthons needed in the preparation of various pharmaceuticals. However, it is incapable of asymmetric reduction when applied to bulky ketones. Recently, mutations at two key residues A85 and I86 were shown to be crucial for reshaping the substrate binding pocket. Increased flexibility of the active site loop appears to be beneficial in the directed evolution of TbSADH towards difficult-to-reduce ketones.

Methods

Using the reported mutant A85G/I86A as template, double-code saturation mutagenesis (DCSM) was applied at selected residues lining the substrate binding pocket with a 2-membered reduced amino acid alphabet.

Results and conclusions

The mutant A85G/I86A was first tested for activity in the reaction of the model substrate (4-chlorophenyl)-(pyridin-2-yl)methanone, which showed a total turnover number (TTN) of 3071. In order to further improve the turnovers, a small and smart mutant library covering a set of mutations at Q101, W110, L294, and C295 was created. Eventually, a triple-mutant A85G/I86A/Q101A was identified to be a superior catalyst that gave S-selective product with 99% ee and 6555 TTN. Docking computations explain the source of enhanced activity. Some of the best variants are also excellent catalysts in the reduction of other difficult-to-reduce ketones.

Keywords

Directed evolution / Conformational dynamics / Alcohol dehydrogenase / Activity / Enantioselectivity

References

[1] Agudo R, Roiban GD, Reetz MT. Induced axial chirality in biocatalytic asymmetric ketone reduction. J Am Chem Soc, 2013, 135(5): 1665-1668.

[2] Arnold FH. Directed evolution: bringing new chemistry to life. Angew Chem Int Ed Engl, 2018, 57(16): 4143-4148.

[3] Barouh V, Dall H, Patel D, Hite G. Stereochemical aspects of antihistamine action. 4. absolute configuration of carbinoxamine antipodes. J Med Chem, 1971, 14(9): 834-836.

[4] Bommarius AS. Biocatalysis: a status report. Annu Rev Chem Biomol Eng, 2015, 6: 319-345.

[5] Combs SA, Deluca SL, Deluca SH, Lemmon GH, Nannemann DP, Nguyen ED, Willis JR, Sheehan JH, Meiler J. Small-molecule ligand docking into comparative models with Rosetta. Nat Protoc, 2013, 8(7): 1277-1298.

[6] Corey EJ, Helal CJ. Reduction of carbonyl compounds with chiral oxazaborolidine catalysts: a new paradigm for enantioselective catalysis and a powerful new synthetic method. Angew Chem Int Ed Engl, 1998, 37(15): 1986-2012.

[7] Denard CA, Ren H, Zhao H. Improving and repurposing biocatalysts via directed evolution. Curr Opin Chem Biol, 2015, 25: 55-64.

[8] Garcia-Urdiales E, Alfonso I, Gotor V. Enantioselective enzymatic desymmetrizations in organic synthesis. Chem Rev, 2005, 105(1): 313-354.

[9] Hagen J. Industrial catalysis: a practical approach, 2006, New York: Wiley.

[10] Heiss C, Laivenieks M, Zeikus JG, Phillips RS. Mutation of cysteine-295 to alanine in secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus affects the enantioselectivity and substrate specificity of ketone reductions. Bioorg Med Chem, 2001, 9(7): 1659-1666.

[11] Huisman GW, Liang J, Krebber A. Practical chiral alcohol manufacture using ketoreductases. Curr Opin Chem Biol, 2010, 14(2): 122-129.

[12] Keinan E, Hafeli EK, Seth KK, Lamed R. Thermostable enzymes in organic-synthesis. 2. asymmetric reduction of ketones with alcohol-dehydrogenase from Thermoanaerobium-brockii. J Am Chem Soc, 1986, 108(1): 162-169.

[13] Kida T, Fujii A, Sakai O, Iemura M, Atsumi I, Wada T, Sakaki H. Bepotastine besilate, a highly selective histamine H(1) receptor antagonist, suppresses vascular hyperpermeability and eosinophil recruitment in in vitro and in vivo experimental allergic conjunctivitis models. Exp Eye Res, 2010, 91(1): 85-91.

[14] Korkhin Y, Kalb AJ, Peretz M, Bogin O, Burstein Y, Frolow F. NADP-dependent bacterial alcohol dehydrogenases: crystal structure, cofactor-binding and cofactor specificity of the ADHs of Clostridium beijerinckii and Thermoanaerobacter brockii. J Mol Biol, 1998, 278(5): 967-981.

[15] Li G, Maria-Solano MA, Romero-Rivera A, Osuna S, Reetz MT. Inducing high activity of a thermophilic enzyme at ambient temperatures by directed evolution. Chem Commun, 2017, 53(68): 9454-9457.

[16] Liang C, Nie Y, Mu X, Xu Y. Gene mining-based identification of aldo-keto reductases for highly stereoselective reduction of bulky ketones. Bioresour Bioprocess, 2018, 5: 33.

[17] Liu B, Qu G, Li J-K, Fan W, Ma J-A, Xu Y, Nie Y, Sun Z. Conformational dynamics-guided loop engineering of an alcohol dehydrogenase: capture, turnover and enantioselective transformation of difficult-to-reduce ketones. Adv Synth Catal, 2019

[18] Maclaren WR, Bruff WC, Eisenberg BC, Weiner H, Martin WH. A clinical comparison of carbinoxamine maleate, tripelennamine hydrochloride, and bromodiphenhydramine hydrochloride in treating allergic symptoms. Ann Allergy, 1955, 13(3): 307-312.

[19] Moa S, Himo F. Quantum chemical study of mechanism and stereoselectivity of secondary alcohol dehydrogenase. J Inorg Biochem, 2017, 175: 259-266.

[20] Morris RH. Asymmetric hydrogenation, transfer hydrogenation and hydrosilylation of ketones catalyzed by iron complexes. Chem Soc Rev, 2009, 38(8): 2282-2291.

[21] Musa MM, Lott N, Laivenieks M, Watanabe L, Vieille C, Phillips RS. A single point mutation reverses the enantiopreference of Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase. ChemCatChem, 2009, 1(1): 89-93.

[22] Nealon CM, Musa MM, Patel JM, Phillips RS. Controlling substrate specificity and stereospecificity of alcohol dehydrogenases. ACS Catal, 2015, 5(4): 2100-2114.

[23] Nie Y, Wang S, Xu Y, Luo S, Zhao Y, Xiao R, Montelione GT, Hunt JF, Szyperski T. Enzyme engineering based on X-ray structures and kinetic profiling of substrate libraries: alcohol dehydrogenases for stereospecific synthesis of a broad range of chiral alcohols. ACS Catal, 2018, 8(6): 5145-5152.

[24] Noyori R. Asymmetric catalysis: science and opportunities (Nobel lecture). Angew Chem Int Ed Engl, 2002, 41(12): 2008-2022.

[25] Qu G, Lonsdale R, Yao P, Li G, Liu B, Reetz MT, Sun Z. Methodology development in directed evolution: exploring options when applying triple-code saturation mutagenesis. ChemBioChem, 2018, 19(3): 239-246.

[26] Reetz MT. Laboratory evolution of stereoselective enzymes: a prolific source of catalysts for asymmetric reactions. Angew Chem Int Ed Engl, 2011, 50(1): 138-174.

[27] Reetz MT. Biocatalysis in organic chemistry and biotechnology: past, present, and future. J Am Chem Soc, 2013, 135(34): 12480-12496.

[28] Reetz MT. The importance of additive and non-additive mutational effects in protein engineering. Angew Chem Int Ed Engl, 2013, 52(10): 2658-2666.

[29] Reetz MT. Alcalde M. Recent advances in directed evolution of stereoselective enzymes. In directed enzyme evolution: advances and applications, 2017, Stuttgart: Springer, 69-99.

[30] Reetz MT, Carballeira JD. Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat Protoc, 2007, 2(4): 891-903.

[31] Reetz MT, Wang LW, Bocola M. Directed evolution of enantioselective enzymes: iterative cycles of CASTing for probing protein-sequence space. Angew Chem Int Ed Engl, 2006, 45(8): 1236-1241.

[32] Reetz MT, Prasad S, Carballeira JD, Gumulya Y, Bocola M. Iterative saturation mutagenesis accelerates laboratory evolution of enzyme stereoselectivity: rigorous comparison with traditional methods. J Am Chem Soc, 2010, 132(26): 9144-9152.

[33] Sandoval CA, Li Y, Ding K, Noyori R. The hydrogenation/transfer hydrogenation network in asymmetric reduction of ketones catalyzed by [RuCl2(binap)(pica)] complexes. Chem Asian J, 2008, 3(10): 1801-1810.

[34] Schrödinger Suite 2015-1 (2015) LLC, New York

[35] Sheldon RA, Pereira PC. Biocatalysis engineering: the big picture. Chem Soc Rev, 2017, 46(10): 2678-2691.

[36] Sun Z, Lonsdale R, Ilie A, Li G, Zhou J, Reetz MT. Catalytic asymmetric reduction of difficult-to-reduce ketones: triple-code saturation mutagenesis of an alcohol dehydrogenase. ACS Catal, 2016, 6(3): 1598-1605.

[37] Sun Z, Li G, Ilie A, Reetz MT. Exploring the substrate scope of mutants derived from the robust alcohol dehydrogenase TbSADH. Tetrahedron Lett, 2016, 57(32): 3648-3651.

[38] Sun Z, Lonsdale R, Li G, Reetz MT. Comparing different strategies in directed evolution of enzyme stereoselectivity: single-versus double-code saturation mutagenesis. ChemBioChem, 2016, 17(19): 1865-1872.

[39] Sun Z, Liu Q, Qu G, Feng Y, Reetz MT. The utility of B-factors in protein science: interpreting rigidity, flexibility and internal motion and engineering thermostability. Chem Rev, 2019, 119: 1626-1665.

[40] Takahashi H, Ishida-Yamamoto A, Iizuka H. Effects of bepotastine, cetirizine, fexofenadine, and olopatadine on histamine-induced wheal- and flare-response, sedation, and psychomotor performance. Clin Exp Dermatol, 2004, 29(5): 526-532.

[41] Xu G, Wang Y, Tang M, Zhou J, Zhao J, Han R, Ni Y. Hydroclassified combinatorial saturation mutagenesis: reshaping substrate binding pockets of KpADH for enantioselective reduction of bulky–bulky ketones. ACS Catal, 2018, 8(9): 8336-8345.