CONTROL OF POLYHYDROXYALKANOATE SYNTHESIS IN AZOTOBACTER-VINELANDII STRAIN UWD

To better understand the reasons for the hyper-accumulation of polyhyd roxyalkanoate (PHA) in mutant Azotobacter vinelandii UWD, the kinetic properties of 3-ketothiolase, acetoacetyl-CoA reductase, and beta-hydr oxybutyrate dehydrogenase were examined. The regulation of the condens ation of acetyl-CoA mediated by 3-ketothiolase was normal, in that it was negatively regulated by free CoA, but inhibition was overcome by h igher concentrations of acetyl-CoA. Acetoacetyl-CoA from this reaction was reduced to 3-hydroxybutyryl-CoA by an NADPH-specific acetoacetyl- CoA reductase. This enzyme also reduced 3-ketovaleryl-CoA derived from the beta-oxidation of C-5, C-7 or C-9 n-alkanoates, but at only 16% o f the rate found with the C-4-substrate. The acetoacetyl-CoA reductase was determined to be an allosteric enzyme that bound NADPH and acetoa cetyl-CoA at multiple binding sites in a general hybrid Ping-Pong rand om mechanism. The enzyme was negatively regulated by acetoacetyl-CoA, but this was overcome at high concentrations of NADPH. The activity of pyridine nucleotide transhydrogenase was determined to be important f or the conversion of NADH in these mutant cells to NADPH and for decre asing the availability of NADP(+), which was a negative regulator of t he acetoacetyl-CoA reductase. The combination of high acetoacetyl-CoA, the UWD mutation, transhydrogenase activity, and high NADPH appeared to be the conditions promoting PHA formation by strain UWD during acti ve growth on glucose. Degradation of PHA in strain UWD did not appear to be regulated at the level of beta-hydroxybutyrate dehydrogenase. Th is enzyme was unaffected by NADH, was inhibited only 13% by pyruvate a nd its activity was enhanced by NADPH. The thiolysis of acetoacetyl-Co A also was unusual, in that 3-ketothiolase was not inhibited by acetoa cetyl-CoA, but free CoA was a competitive inhibitor in a bireactant Pi ng-Pong mechanism. This inhibition was overcome by higher concentratio ns of the normal first substrate, acetoacetyl-CoA. Thus a single thiol ase was used for the condensation of acetyl-CoA and the thiolysis of a cetoacetyl-CoA, derived from PHA depolymerization or from the beta-oxi dation of n-alkanoates.