Crystallographic analysis of a novel aldo‐keto reductase from Thermotoga maritima in complex with NADP+. Issue 7 (1st July 2015)

• Record Type: Journal Article
• Authors: Hou, Hai; Li, Ruiying; Wang, Xiaoyan; Yuan, Zhen; Liu, Xuemeng; Chen, Zhenmin; Xu, Xiaoling
• Is Part Of: Acta crystallographica. Volume 71:Issue 7(2015:Jul.)
• Page Start: 847
• Page End: 855
• Publication Date: 2015-07-01
• Subjects: Crystallography -- Periodicals; Crystals -- Periodicals; 548
• DOI: 10.1107/S2053230X15009735
• Languages: English
• ISSNs: 2053-230X

Abstract

<abstract abstract-type="main" xml:lang="en"> <title> <x xml:space="preserve">Abstract</x> </title> <p>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 <italic>Thermotoga maritima</italic> 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 <italic>Escherichia coli</italic> BL21(DE3) cells with an N‐terminal His₆ tag 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 group <italic>P</italic>3₁21, 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.</p> </abstract>