Resolution of the fatty acid reductase from Photobacterium phosphoreum into acyl protein synthetase and acyl-CoA reductase activities. Evidence for an enzyme complex.

The biosynthesis of long chain aliphatic aldehydes for light emission in luminescent bacteria is catalyzed by a fatty acid reductase in a reaction dependent on ATP and NADPH. Evidence has now been obtained which demonstrates that this reaction consists of two distinct steps: 1) activation of fatty acid and 2) reduction of a fatty acyl intermediate to aldehyde. Fatty acid reductase catalyzed the ATP-dependent incorporation of [3H]tetradecanoic acid into material insoluble in chloroform/methanol/acetic acid (3:6:1) (designated as acyl protein synthetase activity) as well as the reduction of tetradecanoyl-CoA to fatty aldehyde in a reaction dependent on NADPH (acyl-CoA reductase activity). Both activities co-migrated with the fatty acid reductase on anion exchange chromatography and gel filtration. Dye-ligand chromatography on Cibacron blue-Sepharose, however, resolved the fatty acid reductase into its two functional components with the acyl protein synthetase activity being selectively adsorbed on this gel and eluted by a high concentration of sodium thiocyanate. Patty acid reductase activity could only be restored by mixing the two enzyme components. Both the molecular weight and the kinetic properties of the acyl protein synthetase were altered after resolution from the acyl-CoA reductase. The acyl protein product could be converted into a form soluble in organic solvents by treatment with hydroxylamine and eluted on gel filtration at the position of the synthetase indicating that it represented a covalent acyl protein intermediate of the enzyme. Analysis of the nucleotide products after incubation of the enzyme with [CX-~~PIATP and fatty acid showed that ATP was cleaved to AMP during fatty acid activation. These results provide evidence that the fatty acid reductase is an enzyme complex containing at least two functional components, a synthetase involved in activation of the fatty acid to a fatty acyl intermediate with cleavage of ATP to AMP and a reductase that reduces the activated intermediate to aldehyde in an NADPH-dependent step.