Biochemical studies of three Saccharomyces cerevisiae acyl-CoA synthetases, Faa1p, Faa2p, and Faa3p

The efficiency and specificity of protein N-myristoylation appear to be influenced by the availability of myristoyl-CoA and other potential acyl-CoA substrate of myristoyl-CoA:protein N-myristoyltransferase. Recent studies have revealed that Saccharomyces cerevisiae contains at least three acyl-CoA synthetase genes (FAA for fatty acid activation). We have expressed Faa1p, Faa2p, and Faa3p in a strain of Escherichia coli that lacks its own endogenous acyl-CoA synthetase (FadD). Each S. cerevisiae acyl-CoA synthetase contained a carboxyl-terminal His tag so that it could be purified to homogeneity in a single step using nickel chelate affinity chromatography. In vitro assays of C3:0-C24:0 fatty acids indicate that Faa1p prefers C12:0- C16:0, with myristic and pentadecanoic acid (C15:0) having the highest activities. Faa2p can accommodate a wider range of acyl chain lengths: C9:0- C13:0 are preferred and have equivalent activities, although C7:0-C17:0 fatty acids are tolerated as substrates with no greater than a 2-fold variation in specific activity. The myristoyl-CoA synthetase activities of Faa1p and Faa2p are 2 orders of magnitude greater than that of Faa3p in vitro. Faa3p has a preference for C₁₆ and C₁₈ fatty acids with a cis-double bond at C-9-C- 10. The temperature optimum for Faa1p is 30 °C, while Faa2p and Faa3p have the greatest activities at 25 °C. These in vitro observations were confirmed using two in vivo assays: (i) measurement of the ability of each S. cerevisiae acyl-CoA synthetase to direct the incorporation of exogenously derived tritiated myristate, palmitate, or oleate into cellular phospholipids produced in a fadD^- strain of E. coli during exponential growth at 24 or 37 °C and (ii) measurement of the incorporation of [³H]myristate into a yeast N-myristoylprotein coexpressed with NmtIp and Faa1p, Faa2p, or Faa3p in the fadD^- strain.