Crystallization and preliminary X-ray analysis of 5-keto-D-gluconate reductase from Gluconobacter suboxydans IFO12528 complexed with 5-keto-D-gluconate and NADPH.

ABSTRACT: NADPH-dependent 5-keto-D-gluconate reductase from Gluconobacter suboxydans IFO12528 (5KGR) catalyzes oxidoreduction between 5-keto-D-gluconate and D-gluconate with high specificity. 5KGR was expressed in Escherichia coli, purified and crystallized with 5-keto-D-gluconate and NADPH using the sitting-drop vapour-diffusion method at 288?K. A crystal of the 5KGR-NADPH complex was obtained using reservoir solution containing PEG 4000 as a precipitant and diffracted X-rays to 1.75?Å resolution. The crystal of the complex belonged to space group P4(2)2(1)2, with unit-cell parameters a=b=128.6, c=62.9?Å. A crystal of the 5KGR-NADPH-5-keto-D-gluconate complex was prepared by soaking the 5KGR-NADPH complex crystal in reservoir solution supplemented with 100?mM 5-keto-D-gluconate and 10?mM NADPH for 20?min and diffracted X-rays to 2.26?Å resolution. The crystal of the ternary complex belonged to space group P4(2)2(1)2, with unit-cell parameters a=b=128.7, c=62.5?Å. Both crystals contained two molecules in the asymmetric unit.

SUBMITTER: Kubota K 

PROVIDER: S-EPMC2998383 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature