Crystallographic analysis of a novel aldo-keto reductase fromThermotoga maritimain 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 fromThermotoga maritimawas 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 inEscherichia coliBL21(DE3) cells with an N-terminal His6tag and was purified by Ni2+-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 groupP3121, 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.