title: Purification, crystallization and quaternary structure analysis of a glycerol dehydrogenase S305C mutant from Bacillus stearothermophilus.
abstract: Bacillus stearothermophilus glycerol dehydrogenase (GlyDH) is a 39.5 kDa molecular weight metalloenzyme which catalyzes the oxidation of glycerol to dihydroxyacetone with the concomitant reduction of NAD(+) to NADH. Despite its classification as a member of the 'iron-containing' polyol dehydrogenase family, studies on recombinant B. stearothermophilus GlyDH have shown this enzyme to be Zn(2+)-dependent. Crystals of a S305C GlyDH mutant were obtained by the hanging-drop vapour-diffusion method, using ammonium sulfate and PEG 400 as precipitating agents, in the presence and absence of NAD(+). The crystals belong to space group I422, with approximate unit-cell parameters a = b = 105, c = 149 A and one subunit in the asymmetric unit, corresponding to a packing density of 2.6 A(3) Da(-1). The crystals diffract X-rays to at least 1.8 A resolution on a synchrotron-radiation source. Determination of the structure will provide insights into the key determinations of catalytic activity of this class of enzymes, for which no structures are currently available.
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, England.
authors.forename: J
authors.initials: J
authors.lastname: Burke
authors.forename: S N
authors.initials: SN
authors.lastname: Ruzheinikov
authors.forename: S
authors.initials: S
authors.lastname: Sedelnikova
authors.forename: P J
authors.initials: PJ
authors.lastname: Baker
authors.forename: D
authors.initials: D
authors.lastname: Holmes
authors.forename: N M
authors.initials: NM
authors.lastname: Muir
authors.forename: M G
authors.initials: MG
authors.lastname: Gore
authors.forename: D W
authors.initials: DW
authors.lastname: Rice
mesh_terms: D003460:Crystallization; D018360:Crystallography, X-Ray; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D008855:Microscopy, Electron, Scanning; D016296:Mutagenesis; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure
chemical_list: D013401:Sugar Alcohol Dehydrogenases; C020672:glycerol dehydrogenase