Control of polyhydroxyalkanoate synthesis in Azotobacter vinelandii strain UWD

Microbiology, University of Alberta, Edmonton, Alberta T6G 2E9, Canada polyhydroxyalkanoate (PHA) in mutant Azotobacter vinelandii UWD, the kinetic properties of 3-ketothiolase, acetoacetyl-CoA reductase, and /3hydroxybutyrate dehydrogenase were examined. The regulatiorn of the condensation of acetyl-CoA mediated by 3-ketothiolase was narmal, in that it was negatively regulated by free CoA, but inhibition was overeome by higher concentrations of acetyl-CoA. Acetoacetyl-CoA from this reaction was reduced to 3-hydroxybutyryl-CoA by an NADPH-specif ic acetoacetyl-CoA reductase. This enzyme also reduced 3-ketovaleryl-CoA derived from the /?-oxidation of C, C, or C, n-alkanoates, but at only 16% of the rate found with the C,-substrate. The acetoacetyl-CoA reductase was determined to be an allosteric enzyme that bound NADPH and acetoacetyl-CoA at multiple binding sites irl a general hybrid Ping-Pong random mechanism. The enzyme was negatively regulaqed by acetoacetyl-CoA, but th is was overcome at high concentrations of NADPH, The activity of pyridine nucleotide transhydrogenase was determined to be important for the conversion of NADH in these mutant cells t o NADPH and for decreasing the availability of NADP+, which was a negative regulator of the acetoacetyl-CoA reductase. The combination of high acetoacetyl-CoA, the UWD mutation, transhydrogenase activity, and high NADPH appeared t o be the conditions promoting PHA formation by strain UWD during active growth on glucose. Degradation of PHA in strain UWD did not appear to be regulated at the level of /3-hydroxybutyrate dehydrogenase. This enzyme was unaffected by NADH, was inhibited only 13% by pyruvate and its activity was enhanced by NADPH. The thiolysis of acetoacetyl-CoA also was unusual, in that 3ketothiolase was not inhibited by acetoacetyl-CoA, but free CoA was a competitive inhibitor in a bireactant Ping-Pong mechanism. This inhibition was overcome by higher concentrations of the normal first substrate, acetoacetylCoA. Thus a single thiolase was used for the condensation of acetyl-CoA and the thiolysis of acetoacetyl-CoA, derived from PHA depolymerization or from the /?-oxidation of n-alkanoates.