title: Crystallographic analysis of a novel aldo-keto reductase from Thermotoga maritima in complex with NADP⁺.
abstract: Aldo-keto reductases (AKRs) are a superfamily of NAD(P)H-dependent oxidoreductases that catalyse the asymmetric reduction of aldehydes and ketones to chiral alcohols in various organisms. The novel aldo-keto reductase Tm1743 from Thermotoga maritima was identified to have a broad substrate specificity and high thermostability, serving as an important enzyme in biocatalysis and fine-chemical synthesis. In this study, Tm1743 was overexpressed in Escherichia coli BL21(DE3) cells with an N-terminal His6 tag and was purified by Ni(2+)-chelating affinity and size-exclusion chromatography. Purified recombinant enzyme was incubated with its cofactor NADP(+) and its substrate ethyl 2-oxo-4-phenylbutyrate (EOPB) for crystallization. Two X-ray diffraction data sets were collected at 2.0 and 1.7 Å resolution from dodecahedral crystals grown from samples containing Tm1743-NADP(+)-EOPB and Tm1743-NADP(+), respectively. Both crystals belonged to space group P3121, with similar unit-cell parameters. However, in the refined structure model only NADP(+) was observed in the active site of the full-length Tm1743 enzyme. Degradation of the N-terminal vector-derived amino acids during crystallization was confirmed by Western blot and mass-spectrometric analyses.
authors.affiliation: Institute of Ageing Research, School of Medicine, Hangzhou Normal University, Hangzhou 311121, People's Republic of China.
authors.forename: Hai
authors.initials: H
authors.lastname: Hou
authors.affiliation: Institute of Ageing Research, School of Medicine, Hangzhou Normal University, Hangzhou 311121, People's Republic of China.
authors.forename: Ruiying
authors.initials: R
authors.lastname: Li
authors.affiliation: Institute of Ageing Research, School of Medicine, Hangzhou Normal University, Hangzhou 311121, People's Republic of China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Wang
authors.affiliation: Institute of Ageing Research, School of Medicine, Hangzhou Normal University, Hangzhou 311121, People's Republic of China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Yuan
authors.affiliation: Institute of Ageing Research, School of Medicine, Hangzhou Normal University, Hangzhou 311121, People's Republic of China.
authors.forename: Xuemeng
authors.initials: X
authors.lastname: Liu
authors.affiliation: Laboratory of Biocatalysis, Hangzhou Normal University, Hangzhou 311121, People's Republic of China.
authors.forename: Zhenmin
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Institute of Ageing Research, School of Medicine, Hangzhou Normal University, Hangzhou 311121, People's Republic of China.
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Xu
mesh_terms: D000449:Aldehyde Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000074408:Aldo-Keto Reductases; D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray / Q000379:methods; D008969:Molecular Sequence Data; D009249:NADP / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017433:Protein Structure, Secondary; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics
chemical_list: D009249:NADP; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase
keywords: Thermotoga maritima; aldo-keto reductases; thermostable