Studies on the glycerol dehydrogenase from Bacillus stearothermophilus

Glycerol dehydrogenase (GDH) from B.stearothermophilus being an ND+/NADH dependent glycerol 2 : oxidoreductase has the classification E.C.1.1.1.6. The enzyme is a tetramer of identical 42 kDa subunits, requiring the presence of a single bound Zn2+ion per subunit for activity. The enzyme has been shown to be able only to bind its substrate, glycerol, when in the metallo form. The coenzyme NADH was found to bind to both the metal-depleted and metallo forms of the enzyme. Removal of the metal ion from the enzyme results in loss of activity which can be regenerated by the addition of a variety of divalent metal ions. Nickel ions were studied in particular and the on rate K+ and the off rate K- for nickel were determined. The interaction between the enzyme and its coenzyme was studied by performing fluorescence and kinetic experiments using coenzyme analogues. By using the different analogues, those portions of the coenzyme that were necessary for productive binding were determined. The results fitted with the general pattern reported for other dehydrogenases, which was expected in view of the exceedingly similar NAD binding sites in all these enzymes. Peptides generated by various cleavages, both enzymic and chemical, of GDH were isolated by reverse phase HPLC and sequenced on an Applied Biosystems pulsed liquid protein sequencer. Back translation of some of these peptides provided information for the synthesis of oligonucleotide probes specific for the gdh gene. Antisera raised against the whole protein and a specific peptide were used in a number of experiments to investigate the possibility of isolating the gdh gene by immunological methods. The anti-peptide antiserum in particular produced encouraging results to this end. Gene libraries of B.stearothermophilus DNA were successfully produced in both bacteriophage and plasmid vectors. In situ colony by hybridisation experiments led to the isolation of a clone which might have contained part of the gdh gene. Once the nucleotide sequence of this clone had been fully determined, it was found not to contain the gdh gene.