PURIFICATION OF 1,3-PROPANEDIOL DEHYDROGENASE FROM CITROBACTER-FREUNDII AND CLONING, SEQUENCING, AND OVEREXPRESSION OF THE CORRESPONDING GENE IN ESCHERICHIA-COLI

1,3-Propanediol dehydrogenase (EC 1.1.1.202) was purified to homogenei ty from Citrobacter freundii grown anaerobically on glycerol in contin uous culture. The enzyme is an octamer of a polypeptide of 43,400 Da. When tested as a dehydrogenase, the enzyme was most active with substr ates containing two primary alcohol groups separated by one or two car bon atoms. In the physiological direction, 3-hydroxypropionaldehyde,va s the preferred substrate. The apparent K-m values of the enzyme for 3 -hydroxypropionaldehyde and NADH were 140 and 33 mu M, respectively. T he enzyme was inhibited by chelators of divalent cations but could be reactivated by the addition of Fe2+. The dhaT gene, encoding the 1,3-p ropanediol dehydrogenase, was cloned, and its nucleotide sequence (1,1 64 bp) was determined. The deduced dhaT gene product (387 amino acids, 41,324 Da) showed a high level of similarity to a novel family (type III) of alcohol dehydrogenases. The dhaT gene was overexpressed in Esc herichia coli 274-fold by using the T7 RNA polymerase/promoter system.