Cloning, sequencing, and overexpression of the genes encoding coenzyme B-12-dependent glycerol dehydratase of Citrobacter freundii

The genes encoding coenzyme B-12-dependent glycerol dehydratase of Citrobacter freundii were cloned and overexpressed in Escherichia coli. The B-12-free enzyme was purified to homogeneity. It consists of three types of subunits whose N-terminal sequences are in accordance with those deduced from the open reading frames dhaB, dhaC, and dhaE, coding for subunits of 60,433 (alpha), 21,487 (beta), and 16,121 (gamma) Da, respectively, The enzyme complex has the composition alpha(2) beta(2) gamma(2). Amino acid alignments with the subunits of the recently sequenced diol dehydratase of Klebsiella oxytoca (T. Tohimatsu, T. Hara, M. Sakaguchi, Y. Kishimoto, Y. Wada, M. Isoda, T. Sakai, and T. Toraya, J. Biol. Chem. 270:7142-7148, 1995) revealed identities between 51.8 and 70.9%.