Engineering d-Lactate Dehydrogenase from Pediococcus acidilactici for Improved Activity on 2-Hydroxy Acids with Bulky C3 Functional Group
Engineering d-lactic acid dehydrogenases for higher activity on various 2-oxo acids is important for the synthesis of 2-hydroxy acids that can be utilized in a wide range of industrial fields including the production of biopolymers, pharmaceuticals, and cosmetic compounds. Although there are many d-lactate dehydrogenases (d-LDH) available from a diverse range of sources, there is a lack of biocatalysts with high activities for 2-oxo acids with large functional group at C3. In this study, the d-LDH from Pediococcus acidilactici was rationally designed and further engineered by controlling the intermolecular interactions between substrates and the surrounding residues via analysis of the active site structure of d-LDH. As a result, Y51L mutant with the catalytic efficiency on phenylpyruvate of 2200 s−1 mM−1 and Y51F mutant on 2-oxobutryate and 3-methyl-2-oxobutyrate of 37.2 and 23.2 s−1 mM−1 were found, which were 138-, 8.5-, and 26-fold increases than the wild type on the substrates, respectively. Structural analysis revealed that the distance and the nature of the interactions between the side chain of residue 51 and the substrate C3 substituent group significantly affected the kinetic parameters. Bioconversion of phenyllactate as a practical example of production of the 2-hydroxy acids was investigated, and the Y51F mutant presented the highest productivity in in vitro conversion of D-PLA.
Keywordsd-Lactate dehydrogenase 2-Hydroxy acids Substrate specificity Rational design Computational docking
This research was supported by the Technology Development Program to Solve Climate Changes of the National Research Foundation (NRF) funded by the Ministry of Science and ICT (Grant Number, 2017M1A2A2087630).
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflicts of interest.
Research Involving Human Participants and/or Animals
This article does not contain any studies with human participants performed by any of the authors.
- 1.Taguchi, H., & Ohta, T. (1991). D-lactate dehydrogenase is a member of the D-isomer-specific 2-hydroxyacid dehydrogenase family. Cloning, sequencing, and expression in Escherichia coli of the D-lactate dehydrogenase gene of Lactobacillus plantarum. The Journal of Biological Chemistry, 266(19), 12588–12594.PubMedGoogle Scholar
- 3.Södergård, A., & Stolt, M. (2002). Properties of lactic acid based polymers and their correlation with composition. Progress in Polymer Science, 27(6), 1123–1163.Google Scholar
- 4.Tsuji, H., & Okumura, A. (2010). Polymer Journal, 43, 317.Google Scholar
- 5.Cheong, S., Clomburg, J. M., & Gonzalez, R. (2018). A synthetic pathway for the production of 2-hydroxyisovaleric acid in Escherichia coli. Journal of Industrial Microbiology & Biotechnology, 45(7), 579–588.Google Scholar
- 8.Chaudhari, S. S., & Gokhale, D. V. (2016). Journal of Bacteriology & Mycology, 2, 121–125.Google Scholar
- 9.Tokuda, C., Ishikura, Y., Shigematsu, M., Mutoh, H., Tsuzuki, S., Nakahira, Y., Tamura, Y., Shinoda, T., Arai, K., Takahashi, O., & Taguchi, H. (2003). Conversion of Lactobacillus pentosus d-lactate dehydrogenase to a d-hydroxyisocaproate dehydrogenase through a single amino acid replacement. Journal of Bacteriology, 185(16), 5023–5026.PubMedPubMedCentralGoogle Scholar
- 14.Yu, S., Jiang, H., Jiang, B., & Mu, W. (2012). Characterization of D-lactate dehydrogenase producing D-3-phenyllactic acid from Pediococcus pentosaceus. Bioscience Biotechnology and Biochemistry, 76(4), 853–855.Google Scholar
- 19.Antonyuk, S. V., Strange, R. W., Ellis, M. J., Bessho, Y., Kuramitsu, S., Inoue, Y., Yokoyama, S., & Hasnain, S. S. (2009). Structure of D-lactate dehydrogenase from Aquifex aeolicus complexed with NAD+ and lactic acid (or pyruvate). Acta Crystallographica. Section F, Structural Biology and Crystallization Communications, 65(12), 1209–1213.PubMedPubMedCentralGoogle Scholar
- 22.Dengler, U., Niefind, K., Kiess, M., & Schomburg, D. (1997). Crystal structure of a ternary complex of d -2-hydroxyisocaproate dehydrogenase from Lactobacillus casei , NAD + and 2-oxoisocaproate at 1.9 Å resolution 1 1Edited by R. Huber. Journal of Molecular Biology, 267(3), 640–660.PubMedGoogle Scholar
- 25.Prime. (2018). Schrödinger Release 2018-4, Schrödinger. New York, NY: LLC.Google Scholar
- 27.Pramanik, K., Ghosh, P. K., Ray, S., Sarkar, A., Mitra, S., & Maiti, T. K. (2017). An in silico structural, functional and phylogenetic analysis with three dimensional protein modeling of alkaline phosphatase enzyme of Pseudomonas aeruginosa. Journal, Genetic Engineering & Biotechnology, 15(2), 527–537.Google Scholar
- 28.LigPrep. (2018). Schrödinger Release 2018–4, Schrödinger. New York, NY: LLC.Google Scholar
- 29.Friesner, R. A., Banks, J. L., Murphy, R. B., Halgren, T. A., Klicic, J. J., Mainz, D. T., Repasky, M. P., Knoll, E. H., Shelley, M., Perry, J. K., Shaw, D. E., Francis, P., & Shenkin, P. S. (2004). Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. Journal of Medicinal Chemistry, 47(7), 1739–1749.PubMedGoogle Scholar
- 31.Glide. (2018). Schrödinger Release 2018-4. New York, NY: Schrödinger.Google Scholar
- 35.Luthy, R., Bowie, J. U., & Eisenberg, D. (1992). Assessment of protein models with three-dimensional profiles. Nat., 356(6364), 83–85.Google Scholar
- 39.Kumar, A., Kumar, S., Kumar, A., Sharma, N., Sharma, M., Pal Singh, K., & Rathore, M. (2017). Proc Natl Acad Sci, India, Sect B Biol Sci, 88, 1539–1548.Google Scholar