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.