Glycerol dehydrogenase: Structure, specificity, and mechanism of a family III polyol dehydrogenase

Background: Bacillus stearothermophilus glycerol dehydrogenase (GlyDH) (gly cerol:NAD(+) 2-oxidoreductase, EC 1.1.1.6) catalyzes the oxidation of glyce rol 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 famil y. Despite this sequence similarity, GlyDH shows a strict dependence on zin c for activity. On the basis of this, we propose to rename this group the f amily III metaldependent polyol dehydrogenases. To date, no structural data have been reported for any enzyme in this group. Results: The crystal structure of B. stearothermophilus glycerol dehydrogen ase has been determined at 1.7 Angstrom resolution to provide structural in sights into the mechanistic features of this family. The enzyme has 370 ami no 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 t he binary complexes with NAD(+) and glycerol show that the active site of G lyDH lies in the cleft between the enzyme's two domains, with the catalytic zinc ion playing a role in stabilizing an alkoxide intermediate. In additi on, 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 chemis try shares a common chemical step with that catalyzed by glycerol dehydroge nase, providing a striking example of divergent evolution. Finally, the str ucture suggests that the NAD(+) binding domain of GlyDH may be related to t hat of the classical Rossmann fold by switching the sequence order of the t wo mononucleotide binding folds that make up this domain.