Crystallographic analysis of a novel aldo-keto reductase from Thermotoga maritima in complex with NADP+

In this study, Tm1743 was overexpressed in Escherichia coli 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 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.
Source: Acta Crystallographica Section F - Category: Biochemistry Authors: Tags: aldo-keto reductases thermostable Thermotoga maritima research communications Source Type: research