| Acetyl-CoA carboxylase (ACC) catalyzes the first committed step of fatty acid synthesis. ACC has been postulated to be a major rate-limiting enzyme in this pathway, but supporting data are sparse. I report that ACC is indeed a major rate-limiting enzymatic step in the fatty acid synthetic pathway of E. coli. The genes encoding the four subunits of E. coli ACC were cloned under the control of a bacteriophage T7 promoter in a single plasmid. Upon induction of gene expression the four ACC subunits were overproduced in equimolar amounts resulting in greatly increased ACC activity. The product was confirmed to be malonyl-CoA by chromatographic and enzymatic analyses. Overexpression of ACC gave a 10-fold increase in the rate of total cellular lipid synthesis, and a large increase in free fatty acid production upon coexpression of a modified E. coli thioesterase I. The increase in flux is solely due to increased ACC activity, not a labeling artifact.;Enzymatic regulation of ACC was investigated upon the determination that ACC is rate-limiting for the fatty acid synthesis pathway. I concluded the stringent responses alarmone ppGpp does not effect ACC activity in vivo, while any acyl-ACP tested inhibited the total ACC reaction although not the half-reactions. The inhibition of the acyl-ACPs on ACC activity was determined to be competitive in nature and could be relieved by increased acetyl-CoA concentrations. Long chain acyl-ACPs are the end product of fatty acid synthesis and accumulate upon cessation of phospholipid synthesis.;Preliminary functional assignments for the two subunits in the carboxyltransferase complex have been given. The alpha subunit (AccA) appears to have a structural role in the total ACC complex while the beta subunit (AccD) could contain the carboxyltransferase active site. The above conclusions are based on enzymatic deactivational studies with extracts only overproducing AccBCD with and without PEG (14%) compared to extracts with all four subunits overproduced (AccABCD) for the assignment of the AccA function. Extracts only overproducing AccD contained higher carboxyltransferase activity leading one to conclude the active site for the complex is contained in the AccD subunit. |
