title: NADP+-Preferring D-Lactate Dehydrogenase from Sporolactobacillus inulinus.
abstract: Hydroxy acid dehydrogenases, including l- and d-lactate dehydrogenases (L-LDH and D-LDH), are responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids and extensively used in a wide range of biotechnological applications. A common feature of LDHs is their high specificity for NAD(+) as a cofactor. An LDH that could effectively use NADPH as a coenzyme could be an alternative enzymatic system for regeneration of the oxidized, phosphorylated cofactor. In this study, a d-lactate dehydrogenase from a Sporolactobacillus inulinus strain was found to use both NADH and NADPH with high efficiencies and with a preference for NADPH as its coenzyme, which is different from the coenzyme utilization of all previously reported LDHs. The biochemical properties of the D-LDH enzyme were determined by X-ray crystal structural characterization and in vivo and in vitro enzymatic activity analyses. The residue Asn(174) was demonstrated to be critical for NADPH utilization. Characterization of the biochemical properties of this enzyme will contribute to understanding of the catalytic mechanism and provide referential information for shifting the coenzyme utilization specificity of 2-hydroxyacid dehydrogenases.
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People's Republic of China University of Chinese Academy of Sciences, Beijing, People's Republic of China.
authors.forename: Lingfeng
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Institute of Ageing Research, School of Medicine, Hangzhou Normal University, Hangzhou, People's Republic of China.
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Xu
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People's Republic of China.
authors.forename: Limin
authors.initials: L
authors.lastname: Wang
authors.affiliation: Key Laboratory of Tianjin Radiation and Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, People's Republic of China yub@im.ac.cn donghui@irm-cams.ac.cn.
authors.forename: Hui
authors.initials: H
authors.lastname: Dong
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People's Republic of China yub@im.ac.cn donghui@irm-cams.ac.cn.
authors.forename: Bo
authors.initials: B
authors.lastname: Yu
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, People's Republic of China.
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
mesh_terms: D000595:Amino Acid Sequence; D056449:Bacillales / Q000201:enzymology* / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism*; D018360:Crystallography, X-Ray; D007700:Kinetics; D045022:Lactate Dehydrogenases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
chemical_list: D003067:Coenzymes; D009243:NAD; D009249:NADP; D045022:Lactate Dehydrogenases; C071195:D-lactate dehydrogenase