Purification, crystallization and quaternary structure analysis of a glycerol dehydrogenase S305C mutant from Bacillus stearothermophilus

Burke, J., Ruzheinikov, S.N., Sedelnikova, S., Baker, P.J., Holmes, D., Muir, N.M., Gore, M.G. and Rice, D.W. (2001) Purification, crystallization and quaternary structure analysis of a glycerol dehydrogenase S305C mutant from Bacillus stearothermophilus. Acta Crystallographica Section D: Biological Crystallography, 57 (1), 165-167. (doi:10.1107/S0907444900014918).

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 Zn²⁺-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 Å and one subunit in the asymmetric unit, corresponding to a packing density of 2.6 ų Da^-1. The crystals diffract X-rays to at least 1.8 Å 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.

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