Malonyl-CoA : ACP transacylase from Streptomyces coelicolor has two alternative catalytically active nucleophiles

Fatty acids and polyketides are synthesized by mechanistically and evolutio narily related multienzyme systems. Their carbon chain backbones are synthe sized via repeated decarboxylative condensations of alpha -carboxylated bui lding blocks onto a growing acyl chain. These alpha -carboxylated building blocks are transferred from the corresponding coenzyme A thioesters onto th e phosphopantetheine arm of an acyl carrier protein (ACP) by acyl transfera ses, which operate by a ping-pong mechanism involving an acyl-O-serine inte rmediate. In the course of our studies on the malonyl-CoA:ACP transacylase (MAT) from Streptomyces coelicolor, we observed that an active-site Ser (97 ) --> Ala mutant retains activity as well as the ability to be covalently l abeled by C-14 malonyl-CoA. Here we demonstrate that an alternative, cataly tically competent nucleophile exists in the active site of this enzyme. Nex t to the active-site serine is a histidine residue that is conserved in som e, but not all acyl transferases. The H96A mutant is also active and can be labeled, but an H96A/S97A double mutant is inactive and cannot be labeled. The ability of H96 to form a malonyl-imidazole adduct was confirmed by pro teolysis, followed by radio-HPLC and mass spectrometric analysis of the S97 A mutant enzyme. Kinetic analysis revealed that the k(cat) of the S97A muta nt was within 10-fold that of the wild-type enzyme, whereas the K(M)s of th e two enzymes were comparable. Sequence comparison with the E. coli MAT (wh ose X-ray structure is known) led to the identification of H201 as the puta tive base in the serine-histidine catalytic dyad of the S. coelicolor enzym e. The absence of MAT activity in the H201A mutant and the detection of wea k activity in the H201Q mutant was consistent with this proposal. The impli cations of this unexpected finding are discussed.