Regulation of fatty acid composition of Escherichia coli membrane by FabA and FabZ

Abstract

The role of the two dehydratases in type Ii fatty acid biosynthesis, FabA and FabZ, in unsaturated fatty acid (UFA) biosynthesis was investigated by expressing wild-type and two dominate-negative mutants FabA[H70A, D84A] (FabAdb) and FabZ[H54A, E68A] (FabZdb) to increase or knock down their enzyme activity. Protein expression was varied using an arabinose-inducible promoter and the effects were evaluated by metabolic labeling. Increasing FabZ activity reduced UFA production, whereas the FabZ knock down increased UFA formation. Increasing FabA activity decreased UFA synthesis, whereas expression of the dominant-negative FabAdb decreased UFA synthesis and eventually suppressed all fatty acid synthesis. Thus, the elimination of FabA activity with the dominant negative construct was different that the selective inhibition of UFA production observed when the protein activity was eliminated in the fabA mutant. Virtually all the cellular acyl carrier protein (ACP) was found in complex with FabAdb when cell lysates were analyzed by western blot with anti-FabAdb and anti-ACP antibodies. The ACP was purified from these complexes and electrophoretic analysis and mass spectroscopy identified them as acyl-ACPs with chain lengths between 8 to 12 carbons, with β-hydroxy-decanoyl-ACP, the known substrate for FabA, being the most abundant. These results show that FabAdb blocked fatty acid synthesis by sequestering acyl-ACP from processing by other enzymes in the pathway. (Supported by GM 34496)