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Enzyme Microb Technol. 2019 Jun;125:37-44. doi: 10.1016/j.enzmictec.2019.02.008. Epub 2019 Feb 23.

A novel d-2-hydroxy acid dehydrogenase with high substrate preference for phenylpyruvate originating from lactic acid bacteria: Structural analysis on the substrate specificity.

Author information

1
Program of Bioengineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea. Electronic address: hsl0828@snu.ac.kr.
2
Department of Biochemical Engineering, Gangneung-Wonju National University, 7, Jukheon-gil, Gangneung-si, Gangwon-do, 25457, Republic of Korea. Electronic address: jspark@gwnu.ac.kr.
3
Program of Bioengineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea; School of Chemical and Biological Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea. Electronic address: yjyoo@snu.ac.kr.
4
Department of Biochemical Engineering, Gangneung-Wonju National University, 7, Jukheon-gil, Gangneung-si, Gangwon-do, 25457, Republic of Korea. Electronic address: yjyeon@gwnu.ac.kr.

Abstract

2-Hydroxy acid dehydrogenases (2-HADHs) have been implicated in the synthesis of 2-hydroxy acids from 2-oxo acids that are used in wide areas of industry. d-lactate dehydrogenases (d-LDHs), a subfamily of 2-HADH, have been utilized to this purpose, yet they exhibited relatively low catalytic activity to the 2-oxo acids with large functional groups at C3. In this report, four putative 2-HADHs from Oenococcus oeni, Weissella confusa, Weissella koreensis and Pediococcus claussenii were examined for activity on phenylpyruvate (PPA), a substrate to 3-phenyllactic acid (PLA) with a C3 phenyl group. The 2-HADH from P. claussenii was found to have the highest kcat/Km on PPA with 1,348.03 s-1 mM-1 among the four enzymes with higher substrate preference for PPA than pyruvate. Sequential, structural and mutational analysis of the enzyme revealed that it belonged to the d-LDH family, and phenylalanine at the position 51 was the key residue for the PPA binding to the active site via hydrophobic interaction, whereas in the 2-HADHs from O. oeni and W. confusa the hydrophilic tyrosine undermined the interaction. Because phenyllactate is a potential precursor for pharmaceutical compounds, antibiotics and biopolymers, the enzyme could increase the efficiency of bio-production of valuable chemicals. This study suggests a structural basis for the high substrate preference of the 2-HADH, and further engineering possibilities to synthesize versatile 2-hydroxy acids.

KEYWORDS:

Phenyllactic acid; Structural analysis; Substrate specificity; d-2-hydroxy acid dehydrogenase

PMID:
30885323
DOI:
10.1016/j.enzmictec.2019.02.008
[Indexed for MEDLINE]

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