| Summary: | Background: Bacillus stearothermophilus glycerol dehydrogenase (GlyDH) (glycerol:NAD+ 2-oxidoreductase, EC 1.1.1.6) catalyzes the oxidation of glycerol to dihydroxyacetone (1,3-dihydroxypropanone) with concomitant reduction of NAD+ to NADH. Analysis of the sequence of this enzyme indicates that it is a member of the so-called iron-containing alcohol dehydrogenase family. Despite this sequence similarity, GlyDH shows a strict dependence on zinc for activity. On the basis of this, we propose to rename this group the family III metal-dependent polyol dehydrogenases. To date, no structural data have been reported for any enzyme in this group. Results: The crystal structure of B. stearothermophilus glycerol dehydrogenase has been determined at 1.7 Å resolution to provide structural insights into the mechanistic features of this family. The enzyme has 370 amino acid residues, has a molecular mass of 39.5 kDa, and is a homooctamer in solution. Conclusions: Analysis of the crystal structures of the free enzyme and of the binary complexes with NAD+ and glycerol show that the active site of GlyDH lies in the cleft between the enzyme's two domains, with the catalytic zinc ion playing a role in stabilizing an alkoxide intermediate. In addition, the specificity of this enzyme for a range of diols can be understood, as both hydroxyls of the glycerol form ligands to the enzyme-bound Zn2+ ion at the active site. The structure further reveals a previously unsuspected similarity to dehydroquinate synthase, an enzyme whose more complex chemistry shares a common chemical step with that catalyzed by glycerol dehydrogenase, providing a striking example of divergent evolution. Finally, the structure suggests that the NAD+ binding domain of GlyDH may be related to that of the classical Rossmann fold by switching the sequence order of the two mononucleotide binding folds that make up this domain.
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