The three gldCDE genes from Lactobacillus diolivorans, that encode the three subunits of the glycerol dehydratase, were cloned and the proteins were co-expressed in soluble form in Escherichia coli with added sorbitol and betaine hydrochloride. The purified enzyme exists as a heterohexamer (alpha(2)beta(2)gamma(2)) structure with a native molecular mass of 210 kDa. It requires coenzyme B-12 for catalytic activity and is subject to suicide inactivation by glycerol during catalysis. The enzyme had maximum activity at pH 8.6 and 37 A degrees C. The apparent K (m) values for coenzyme B-12, 1,2-ethanediol, 1,2-propanediol, and glycerol were 1.5 mu M, 10.5 mM, 1.3 mM, and 5.8 mM, respectively. Together, these results indicated that the three genes gldCDE encoding the proteins make up a coenzyme B-12-dependent diol dehydratase and not a glycerol dehydratase.