Insight into the Mechanism of the B12-Independent Glycerol Dehydratase from Clostridium butyricum: Preliminary Biochemical and Structural Characterization

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Abstract
The molecular characterization of a B12-independent glycerol dehydratase from Clostridium butyricum has recently been reported [Raynaud, C., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 5010−5015]. In this work, we have further characterized this system by biochemical and crystallographic methods. Both the glycerol dehydratase (GD) and the GD-activating enzyme (GD-AE) could be purified to homogeneity under aerobic conditions. In this form, both the GD and GD-AE were inactive. A reconstitution procedure, similar to what has been reported for pyruvate formate lyase activating enzyme (PFL-AE), was employed to reconstitute the activity of the GD-AE. Subsequently, the reconstituted GD-AE could be used to reactivate the GD under strictly anaerobic conditions. We also report here the crystal structure of the inactive GD in the native (2.5 Å resolution, Rcryst = 17%, Rfree = 20%), glycerol-bound (1.8 Å resolution, Rcryst = 21%, Rfree = 24%), and 1,2-propanediol-bound (2.4 Å resolution, Rcryst = 20%, Rfree = 24%) forms. The overall fold of the GD monomer was similar to what has been observed for pyruvate formate lyase (PFL) and anaerobic ribonucleotide reductase (ARNR), consisting of a 10-stranded β/α barrel motif. Clear density was observed for both substrates, and a mechanism for the dehydration reaction is presented. This mechanism clearly supports a concerted pathway for migration of the OH group through a cyclic transition state that is stabilized by partial protonation of the migrating OH group. Finally, despite poor alignment (rmsd ∼ 6.8 Å) of the 10 core strands that comprise the barrel structure of the GD and PFL, the C-terminal domains of both proteins align well (rmsd ∼ 0.7 Å) and have structural properties consistent with this being the docking site for the activating enzyme. A single point mutation within this domain, at a strictly conserved arginine residue (R782K) in the GD, resulted in formation of a tight protein−protein complex between the GD and the GD-AE in vivo, thereby supporting this hypothesis.

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