Open AccessCloning, expression, purification and preliminary crystallographic analysis of the short‐chain dehydrogenase enzymes WbmF, WbmG and WbmH from Bordetella bronchiseptica
Author(s) -
Harmer Nicholas J.,
King Jerry D.,
Palmer Colin M.,
Preston Andrew,
Maskell Duncan J.,
Blundell Tom L.
Publication year - 2007
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s174430910703477x
Subject(s) - cofactor , crystallization , dehydrogenase , enzyme , escherichia coli , nucleotide , bordetella , bordetella bronchiseptica , chemistry , molecular replacement , biochemistry , crystallography , stereochemistry , substrate (aquarium) , biology , bacteria , gene , organic chemistry , genetics , ecology , bordetella pertussis
The short‐chain dehydrogenase enzymes WbmF, WbmG and WbmH from Bordetella bronchiseptica were cloned into Escherichia coli expression vectors, overexpressed and purified to homogeneity. Crystals of all three wild‐type enzymes were obtained using vapour‐diffusion crystallization with high‐molecular‐weight PEGs as a primary precipitant at alkaline pH. Some of the crystallization conditions permitted the soaking of crystals with cofactors and nucleotides or nucleotide sugars, which are possible substrate compounds, and further conditions provided co‐complexes of two of the proteins with these compounds. The crystals diffracted to resolutions of between 1.50 and 2.40 Å at synchrotron X‐ray sources. The synchrotron data obtained were sufficient to determine eight structures of the three enzymes in complex with a variety of cofactors and substrate molecules.