Open AccessCrystallization and preliminary X‐ray analysis of 5‐keto‐ d ‐gluconate reductase from Gluconobacter suboxydans IFO12528 complexed with 5‐keto‐ d ‐gluconate and NADPH
Author(s) -
Kubota Keiko,
Miyazono Kenichi,
Nagata Koji,
Toyama Hirohide,
Matsushita Kazunobu,
Tanokura Masaru
Publication year - 2010
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309110043617
Subject(s) - chemistry , reductase , ternary operation , crystallization , ternary complex , escherichia coli , crystallography , crystal structure , crystal (programming language) , stereochemistry , enzyme , biochemistry , organic chemistry , computer science , programming language , gene
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 P 4 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 m M 5‐keto‐ d ‐gluconate and 10 m M NADPH for 20 min and diffracted X‐rays to 2.26 Å resolution. The crystal of the ternary complex belonged to space group P 4 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.