Cloning, expression, purification and preliminary crystallographic analysis of the short-chain dehydrogenase enzymes WbmF, WbmG and WbmH from Bordetella bronchiseptica

Journal Article · Wed Aug 01 04:00:00 EDT 2007 · Acta Crystallographica. Section F

DOI:https://doi.org/10.1107/S174430910703477X· OSTI ID:22360381

King, Jerry D. ^[1]; Palmer, Colin M. ^[1]; Preston, Andrew ^[2]; Maskell, Duncan J. ^[2]; Blundell, Tom L. ^[1]

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1. Department of Biochemistry, 80 Tennis Court Road, Cambridge CB2 1GA (United Kingdom)
2. Department of Veterinary Medicine, Cambridge CB3 0ES (United Kingdom)

The expression, purification, and crystallisation of the short-chain dehydrogenases WbmF, WbmG and WbmH from B. bronchiseptica are described. Native diffraction data to 1.5, 2.0, and 2.2 Å were obtained for the three proteins, together with complexes with nucleotides. 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.

• OSTI ID: 22360381
• Journal Information: Acta Crystallographica. Section F, Journal Name: Acta Crystallographica. Section F Journal Issue: Pt 8 Vol. 63; ISSN ACSFCL; ISSN 1744-3091
• Country of Publication: United Kingdom
• Language: English

Related Subjects

75 CONDENSED MATTER PHYSICS
SUPERCONDUCTIVITY AND SUPERFLUIDITY
CRYSTALLIZATION
CRYSTALS
DIFFRACTION
DIFFUSION
ESCHERICHIA COLI
MOLECULAR WEIGHT
MOLECULES
RESOLUTION
SACCHAROSE
SUBSTRATES
SYNCHROTRONS
VECTORS