BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF THE OXIDATIVE BRANCH OFGLYCEROL UTILIZATION BY CITROBACTER-FREUNDII

Glycerol dehydrogenase (EC 1.1.1.6) and dihydroxyacetone kinase (EC 2. 7.1.29) were purified from Citrobacter freundii. The dehydrogenase is a hexamer of a polypeptide of 43,000 Da. The enzyme exhibited a rather broad substrate specificity, but glycerol was the preferred substrate in the physiological direction. The apparent K(m)s of the enzyme for glycerol and NAD(+) were 1.27 mM and 57 mu M, respectively. The kinase is a dimer of a polypeptide of 57,000 Da. The enzyme was highly speci fic for the substrates dihydroxyacetone and ATP; the apparent K(m)s we re 30 and 70 mu M, respectively. The DNA region which contained the ge nes encoding glycerol dehydrogenase (dhaD) and dihydroxyacetone kinase (dhaK) was cloned and sequenced. Both genes were identified by N-term inal sequence comparison. The deduced dhaD gene product (365 amino aci ds) exhibited high degrees of homology to glycerol dehydrogenases from other organisms and less homology to type III alcohol dehydrogenases, whereas the dhaK gene product (552 amino acids) revealed no significa nt homology to any other protein in the databases. A large gene (dhaR) of 1,929 bp was found downstream from dhaD. The deduced gene product (641 amino acids) showed significant similarities to members of the si gma(54) bacterial enhancer-binding protein family.