Cloning, expression, purification and preliminary crystallographic analysis of the short-chain dehydrogenase enzymes WbmF, WbmG and WbmH from Bordetella bronchiseptica.
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 A 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.
Department of Biochemistry, 80 Tennis Court Road, Cambridge CB2 1GA, England. nic@cryst.bioc.cam.ac.uk
Nicholas J
NJ
Harmer
Jerry D
JD
King
Colin M
CM
Palmer
Andrew
A
Preston
Duncan J
DJ
Maskell
Tom L
TL
Blundell
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000145:classification / Q000235:genetics; D016950:Bordetella bronchiseptica / Q000201:enzymology*; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D010088:Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry* / Q000145:classification / Q000235:genetics*
D001426:Bacterial Proteins; D010088:Oxidoreductases