Abstract
The substrate specificity of 3-hydroxybutyrate dehydrogenase from Alcaligenes faecalis with a non-native substrate, levulinic acid, was studied by analysis of the enzyme-substrate molecular interactions. The relation between structural and kinetic parameters was investigated considering the catalytic mechanism of the enzyme. The effects of key positive mutations (H144L, H144L/W187F) on the catalytic activity of the enzyme were studied by employing a surface analysis of its interatomic contacts between the enzyme and substrate atoms. The results revealed that the alteration of hydrogen bond network and rearrangement of the hydrophobic interactions between the active site and substrate molecule are the key structural basis for the change of the substrate specificity of 3-hydroxybutyrate dehydrogenase toward levulinic acid. With this approach, the structural basis for the substrate specificity of the enzyme could be elucidated in a quantitative manner.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Wohlgemuth, R. (2010) Biocatalysis-key to sustainable industrial chemistry. Curr. Opin. Biotechnol. 21: 713–724.
Privett, H. K., G. Kiss, T. M. Lee, R. Blomberg, R. A. Chica, L. M. Thomas, D. Hilvert, K. N. Houk, and S. L. Mayo (2012) Iterative approach to computational enzyme design. Proc. Natl. Acad. Sci. U S A. 109: 3790–3795.
Bornscheuer, U. T., G. W. Huisman, R. J. Kazlauskas, S. Lutz, J. C. Moore, and K. Robins (2012) Engineering the third wave of biocatalysis. Nature 484: 185–194.
Greenhagen, B. T., P. E. O'Maille, J. P. Noel, and J. Chappell (2006) Identifying and manipulating structural determinates linking catalytic specificities in terpene synthases. Proc. Natl. Acad. Sci. U S A. 103: 9826–9831.
Zhang, K., M. R. Sawaya, D. S. Eisenberg, and J. C. Liao (2008) Expanding metabolism for biosynthesis of nonnatural alcohols. Proc. Natl. Acad. Sci. U S A. 105: 20653–20658.
Savile, C. K., J. M. Janey, E. C. Mundorff, J. C. Moore, S. Tam, W. R. Jarvis, J. C. Colbeck, A. Krebber, F. J. Fleitz, J. Brands, P. N. Devine, G. W. Huisman, and G. J. Hughes (2010) Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Sci. 329: 305–309.
Yeon, Y. J., H. Y. Park, and Y. J. Yoo (2013) Enzymatic reduction of levulinic acid by engineering the substrate specificity of 3-hydroxybutyrate dehydrogenase. Bioresour. Technol. 134: 377–380.
Hoque, M. M., S. Shimizu, E. C. M. Juan, Y. Sato, M. T. Hossain, T. Yamamoto, S. Imamura, K. Suzuki, H. Amano, T. Sekiguchi, M. Tsunoda, and A. Takénaka (2009) Structure of D-3-hydroxybutyrate dehydrogenase prepared in the presence of the substrate D-3-hydroxybutyrate and NAD +. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 65: 331–335.
Rackemann, D. W. and W. O. Doherty (2011) The conversion of lignocellulosics to levulinic acid. Biofuels Bioprod. Biorefin. 5: 198–214.
Gorenflo, V., G. Schmack, R. Vogel, and A. Steinbüchel (2001) Development of a process for the biotechnological large-scale production of 4-hydroxyvalerate-containing polyesters and characterization of their physical and mechanical properties. Biomacromol. 2: 45–57.
Horváth, I. T., H. Mehdi, V. Fábos, L. Boda, and L. T. Mika (2008) γ-Valerolactone-a sustainable liquid for energy and carbon-based chemicals. Green Chem. 10: 238–242.
Hoque, M. M., S. Shimizu, M. T. Hossain, T. Yamamoto, S. Imamura, K. Suzuki, M. Tsunoda, H. Amano, T. Sekiguchi, and A. Takénaka (2008) The structures of Alcaligenes faecalis D-3-hydroxybutyrate dehydrogenase before and after NAD+ and acetate binding suggest a dynamical reaction mechanism as a member of the SDR family. Acta Crystallogr. Sect. D: Biol. Crystallogr. 64: 496–505.
Hoque, M. M., S. Shimizu, E. C. M. Juan, Y. Sato, M. T. Hossain, T. Yamamoto, S. Imamura, K. Suzuki, H. Amano, T. Sekiguchi, M. Tsunoda, and A. Takénaka (2009) Structure of D-3-hydroxybutyrate dehydrogenase prepared in the presence of the substrate D-3-hydroxybutyrate and NAD+. Acta Crystallogr. Sect. F: Struct. Biol. Cryst. Commun. 65: 331–335.
Basner, J. E. and S. D. Schwartz (2004) Donor-acceptor distance and protein promoting vibration coupling to hydride transfer: A possible mechanism for kinetic control in isozymes of human lactate dehydrogenase. J. Phys. Chem. B. 108: 444–451.
Pudney, C. R., L. O. Johannissen, M. J. Sutcliffe, S. Hay, and N. S. Scrutton (2010) Direct analysis of donor-acceptor distance and relationship to isotope effects and the force constant for barrier compression in enzymatic H-tunneling reactions. J. Am. Chem. Soc. 132: 11329–11335.
Stojkovi, V., L. L. Perissinotti, D. Willmer, S. J. Benkovic, and A. Kohen (2012) Effects of the donor-acceptor distance and dynamics on hydride tunneling in the dihydrofolate reductase catalyzed reaction. J. Am. Chem. Soc. 134: 1738–1745.
Vasavada, M., C. E. Carpenter, D. P. Cornforth, and V. Ghorpade (2003) Sodium levulinate and sodium lactate effects on microbial growth and stability of fresh pork and turkey sausages. J. Muscle Foods. 14: 119–129.
Zheng, L., U. Baumann, and J. L. Reymond (2004) An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res. 32: e115.
Kim, S. H., S. Pokhrel, and Y. J. Yoo (2008) Mutation of non-conserved amino acids surrounding catalytic site to shift pH optimum of Bacillus circulans xylanase. J. Mol. Catal. B: Enz. 55: 130–136.
Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72: 248–254.
Clark, M., R. D. Cramer, and N. Van Opdenbosch (1989) Validation of the general purpose tripos 5.2 force field. J. Comput. Chem. 10: 982–1012.
Purcell, W. P. and J. A. Singer (1967) A brief review and table of semiempirical parameters used in the Hueckel molecular orbital method. J. Chem. Eng. Data. 12: 235–246.
Lovell, S. C., J. M. Word, J. S. Richardson, and D. C. Richardson (2000) The penultimate rotamer library. Proteins: Struct. Funct. Bioinf. 40: 389–408.
Jain, A. N. (2003) Surflex: Fully Automatic flexible molecular docking using a molecular similarity-based search engine. J. Med. Chem. 46: 499–511.
Sobolev, V., A. Sorokine, J. Prilusky, E. E. Abola, and M. Edelman (1999) Automated analysis of interatomic contacts in proteins. Bioinformat. 15: 327–332.
Yeon, Y. J., H. J. Park, H. Y. Park, and Y. J. Yoo (2014) Effect of His-tag location on the catalytic activity of 3-hydroxybutyrate dehydrogenase. Biotechnol. Bioproc. Eng. 19: 798–802.
Kamerlin, S. C. L. and A. Warshel (2010) At the dawn of the 21st century: Is dynamics the missing link for understanding enzyme catalysis. Proteins: Struct. Funct. Bioinformat. 78: 1339–1375.
Bhabha, G., J. Lee, D. C. Ekiert, J. Gam, I. A. Wilson, H. J. Dyson, S. J. Benkovic, and P. E. Wright (2011) A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis. Sci. 332: 234–238.
Park, J. C., J. C. Joo, E. S. An, B. K. Song, Y. H. Kim, and Y. J. Yoo (2011) A combined approach of experiments and computational docking simulation to the Coprinus cinereus peroxidasecatalyzed oxidative polymerization of alkyl phenols. Bioresour. Technol. 102: 4901–4904.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Yeon, Y.J., Park, HY., Park, K. et al. Structural basis for the substrate specificity of 3-hydroxybutyrate dehydrogenase. Biotechnol Bioproc E 21, 364–372 (2016). https://doi.org/10.1007/s12257-016-0233-2
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12257-016-0233-2