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

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 Å³ 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.