Alteration of the substrate specificity of the malonyl-CoA/acetyl-CoA:acyl carrier protein S-acyltransferase domain of the multifunctional fatty acid synthase by mutation of a single arginine residue.

The structural basis for the dual specificity of the malonyl-CoA/acetyl-CoA:acyl carrier protein S-acyltransferase associated with the multifunctional animal fatty acid synthase has been investigated by mutagenesis. Arginine 606, which is positionally conserved in the transacylase domains of all multifunctional fatty acid and polyketide synthases, was replaced by alanine or lysine in the context of the isolated transacylase domain, and the mutant proteins were expressed in Escherichia coli. Malonyl transacylase activity of the Arg-606 --> Ala and Arg-606 --> Lys mutant enzymes was reduced by 100- and 10-fold, respectively. In contrast, acetyl transacylase activity was increased 6.6-fold in the Arg-606 --> Ala mutant and 1.7-fold in the Arg-606 --> Lys mutant. Kinetic studies revealed that selectivity of the enzyme for acetyl-CoA was increased >16,000-fold by the Ala mutation and 16-fold by the Lys mutation. Activity toward medium chain length acyl thioesters was also increased >3 orders of magnitude by mutation of Arg-606, so that the Ala-606 enzyme is an effective medium chain length fatty acyl transacylase. These results indicate that Arg-606 plays an important role in the binding of malonyl moieties to the transacylase domain but is not required for binding of acetyl moieties; these results are also consistent with a mechanism whereby interaction between the positively charged guanidinium group of Arg-606 and the free carboxylate anion of the malonyl moiety serves to position this substrate in the active site of the enzyme.