Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Impact of Expanded Small Alkyl-Binding Pocket by Triple Point Mutations on Substrate Specificity of Thermoanaerobacter ethanolicus Secondary Alcohol Dehydrogenase

  • Dwamena, Amos K. (Department of Chemical & Biological Engineering, Hanbat National University) ;
  • Phillips, Robert S. (Department of Chemistry, University of Georgia) ;
  • Kim, Chang Sup (Department of Chemical & Biological Engineering, Hanbat National University)
  • Received : 2018.12.11
  • Accepted : 2019.01.01
  • Published : 2019.03.28
Download PDF
⟨ Previous Next ⟩

Abstract

Site-directed mutagenesis was employed to generate five different triple point mutations in the double mutant (C295A/I86A) of Thermoanaerobacter ethanolicus alcohol dehydrogenase (TeSADH) by computer-aided modeling with the aim of widening the small alkyl-binding pocket. TeSADH engineering enables the enzyme to accept sterically hindered substrates that could not be accepted by the wild-type enzyme. The underline in the mutations highlights the additional point mutation on the double mutant TeSADH introduced in this work. The catalytic efficiency ($k_{cat}/K_M$) of the ${\underline{M151A}}$/C295A/I86A triple TeSADH mutant for acetophenone increased about 4.8-fold higher than that of the double mutant. A 2.4-fold increase in conversion of 3'-methylacetophenone to (R)-1-(3-methylphenyl)-ethanol with a yield of 87% was obtained by using ${\underline{V115A}}$/C295A/I86A mutant in asymmetric reduction. The ${\underline{A85G}}$/C295A/I86A mutant also produced (R)-1-(3-methylphenyl)-ethanol (1.7-fold) from 3'-methylacetophenone and (R)-1-(3-methoxyphenyl)-ethanol (1.2-fold) from 3'-methoxyacetophenone, with improved yield. In terms of thermal stability, the ${\underline{M151A}}$/C295A/I86A and ${\underline{V115A}}$/C295A/I86A mutants significantly increased ${\Delta}T_{1/2}$ by $+6.8^{\circ}C$ and $+2.4^{\circ}C$, respectively, with thermal deactivation constant ($k_d$) close to the wild-type enzyme. The ${\underline{M151A}}$/C295A/I86A mutant reacts optimally at $70^{\circ}C$ with almost 4 times more residual activity than the wild type. Considering broad substrate tolerance and thermal stability together, it would be promising to produce (R)-1-(3-methylphenyl)-ethanol from 3'-methylacetophenone by ${\underline{V115A}}$/C295A/I86A, and (R)-1-phenylethanol from acetophenone by ${\underline{M151A}}$/C295A/I86A mutant, in large-scale bioreduction processes.

Keywords

References

  1. Keinan E, Hafeli EK, Seth KK, Lamed R. 1986. Thermostable enzymes in organic synthesis. 2. Asymmetric reduction of ketones with alcohol dehydrogenase from Thermoanaerobium brockii. J. Am. Chem. Soc. 108: 162-169. https://doi.org/10.1021/ja00261a026
  2. Patel RN. 2008. Synthesis of chiral pharmaceutical intermediates by biocatalysis. Coordin. Chem. Rev. 252: 659-701. https://doi.org/10.1016/j.ccr.2007.10.031
  3. De Smidt O, Du Preez JC, Albertyn J. 2008. The alcohol dehydrogenases of Saccharomyces cerevisiae: a comprehensive review. FEMS Yeast Res. 8: 967-978. https://doi.org/10.1111/j.1567-1364.2008.00387.x
  4. Bolle X, Vinals C, Prozzi D, Paquet JY, Leplae R, Depiereux E, et al. 1995. Identification of residues potentially involved in the interactions between subunits in yeast alcohol dehydrogenases. Eur. J. Biochem. 231: 214-219. https://doi.org/10.1111/j.1432-1033.1995.tb20689.x
  5. Nakamura K, Yamanaka R, Matsuda T, Harada T. 2003. Recent developments in asymmetric reduction of ketones with biocatalysts. Tetrahedron:Asymmetr. 14: 2659-2681. https://doi.org/10.1016/S0957-4166(03)00526-3
  6. Muller M, Wolberg M, Schubert T, Hummel W. 2005. Enzymecatalyzed regio-and enantioselective ketone reductions. Adv. Biochem. Eng. Biotechnol. 92: 261-287.
  7. Matsuda T, Yamanaka R, Nakamura K. 2009. Recent progress in biocatalysis for asymmetric oxidation and reduction. Tetrahedron:Asymmetr. 20: 513-557. https://doi.org/10.1016/j.tetasy.2008.12.035
  8. Musa MM, Phillips RS. 2011. Recent advances in alcohol dehydrogenase-catalyzed asymmetric production of hydrophobic alcohols. Catal. Sci. Technol. 1: 1311-1323. https://doi.org/10.1039/c1cy00160d
  9. Nealon CM, Musa MM, Patel JM, Phillips RS. 2015. Controlling substrate specificity and stereospecificity of alcohol dehydrogenases. ACS Catal. 5: 2100-2114. https://doi.org/10.1021/cs501457v
  10. Bradshaw CW, Hummel W, Wong CH. 1992. Lactobacillus kefir alcohol dehydrogenase: a useful catalyst for synthesis. J. Org. Chem. 57: 1532-1536. https://doi.org/10.1021/jo00031a037
  11. Bradshaw CW, Fu H, Shen GJ, Wong CH. 1992. A Pseudomonas sp. alcohol dehydrogenase with broad substrate specificity and unusual stereospecificity for organic synthesis. J. Org. Chem. 57: 1526-1532. https://doi.org/10.1021/jo00031a036
  12. Yang Z-H, Zeng R, Yang G, Wang Y, Li L-Z, Lv Z-S, et al. 2008. Asymmetric reduction of prochiral ketones to chiral alcohols catalyzed by plants tissue. J. Ind. Microbiol. Biotechnol. 35: 1047-1051. https://doi.org/10.1007/s10295-008-0381-2
  13. Heiss C, Laivenieks M, Zeikus JG, Phillips RS. 2001. 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. 9: 1659-1666. https://doi.org/10.1016/S0968-0896(01)00073-6
  14. Musa MM, Lott N, Laivenieks M, Watanabe L, Vieille C, Phillips RS. 2009. A single point mutation reverses the enantiopreference of Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase. ChemCatchem. 1: 89-93. https://doi.org/10.1002/cctc.200900033
  15. Musa MM, Ziegelmann-Fjeld KI, Vieille C, Zeikus JG, Phillips RS. 2007. Asymmetric reduction and oxidation of aromatic ketones and alcohols using W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus. J. Org. Chem. 72: 30-34. https://doi.org/10.1021/jo0616097
  16. Musa MM, Ziegelmann-Fjeld KI, Vieille C, Phillips RS. 2008. Activity and selectivity of W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus in organic solvents and ionic liquids: mono-and biphasic media. Org. Biomol. Chem. 6: 887-892. https://doi.org/10.1039/b717120j
  17. Maniatis T, Fritsch EF, Sambrook J. 1982. Molecular cloning: a laboratory manual, Cold spring harbor laboratory, Cold Spring Harbor, NY.
  18. Ziegelmann-Fjeld KI, Musa MM, Phillips RS, Zeikus JG, Vieille C. 2007. A Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase mutant derivative highly active and stereoselective on phenylacetone and benzylacetone. Protein Eng. Des. Sel. 20: 47-55. https://doi.org/10.1093/protein/gzl052
  19. Arnold K, Bordoli L, Kopp J, Schwede T. 2006. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22: 195-201. https://doi.org/10.1093/bioinformatics/bti770
  20. Guex N, Peitsch MC. 1997. Swiss-Model and the Swiss-Pdb Viewer: an environment for comparative protein modeling. Electrophoresis 18: 2714-2723. https://doi.org/10.1002/elps.1150181505
  21. DeLano WL. 2002. The PyMOL molecular graphics system. http://www. pymol.org.
  22. Li C, Heatwole J, Soelaiman S, Shoham M. 1999. Crystal structure of a thermophilic alcohol dehydrogenase substrate complex suggests determinants of substrate specificity and thermostability. Proteins 37: 619-627. https://doi.org/10.1002/(SICI)1097-0134(19991201)37:4<619::AID-PROT12>3.0.CO;2-H
  23. Benkert P, Biasini M, Schwede T. 2011. Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics 27: 343-350. https://doi.org/10.1093/bioinformatics/btq662
  24. Goihberg E, Dym O, Tel-Or S, Shimon L, Frolow F, Peretz M, et al. 2008. Thermal stabilization of the protozoan Entamoeba histolytica alcohol dehydrogenase by a single proline substitution. Proteins 72: 711-719. https://doi.org/10.1002/prot.21946
  25. Bogin O, Peretz M, Hacham Y, Burstein Y, Korkhin Y, Frolow F. 1998. Enhanced thermal stability of Clostridium beijerinckii alcohol dehydrogenase after strategic substitution of amino acid residues with prolines from the homologous thermophilic Thermoanaerobacter brockii alcohol dehydrogenase. Protein Sci. 7: 1156-1163. https://doi.org/10.1002/pro.5560070509
  26. Soni S, Desai J, Devi S. 2001. Immobilization of yeast alcohol dehydrogenase by entrapment and covalent binding to polymeric supports. J. Appl. Polym. Sci. 82: 1299-1305. https://doi.org/10.1002/app.1964
  27. Naik HG, Yeniad B, Koning CE, Heise A. 2012. Investigation of asymmetric alcohol dehydrogenase (ADH) reduction of acetophenone derivatives: effect of charge density. Org. Biomol. Chem. 10: 4961-4967. https://doi.org/10.1039/c2ob06870b
  28. Musa MM, Patel JM, Nealon CM, Kim CS, Phillips RS, Karume I. 2015. Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase mutants with improved racemization activity. J. Mol. Catal. B-Enzym. 115: 155-159. https://doi.org/10.1016/j.molcatb.2015.02.012
  29. Rodriguez C, Borzęcka W, Sattler JH, Kroutil W, Lavandera I, Gotor V. 2014. Steric vs. electronic effects in the Lactobacillus brevis ADH-catalyzed bioreduction of ketones. Org. Biomol. Chem. 12: 673-681. https://doi.org/10.1039/C3OB42057D
  30. Burdette DS, Tchernajencko V, Zeikus JG. 2000. Effect of thermal and chemical denaturants on Thermoanaerobacter ethanolicus secondary-alcohol dehydrogenase stability and activity. Enzyme Microb. Technol. 27: 11-18. https://doi.org/10.1016/S0141-0229(00)00192-7
  31. Ganter C, Plueckthun A. 1990. Glycine to alanine substitutions in helixes of glyceraldehyde-3-phosphate dehydrogenase: effects on stability. Biochemistry 29: 9395-9402. https://doi.org/10.1021/bi00492a013
  32. Pace CN, Shirley BA, McNutt M, Gajiwala K. 1996. Forces contributing to the conformational stability of proteins. FASEB J. 10: 75-83. https://doi.org/10.1096/fasebj.10.1.8566551
  33. Korkhin Y, Kalb AJ, Peretz M, Bogin O, Burstein Y, Frolow F. 1999. Oligomeric integrity-the structural key to thermal stability in bacterial alcohol dehydrogenases. Protein Sci. 8: 1241-1249. https://doi.org/10.1110/ps.8.6.1241