Malonyl-CoA:ACP Transacylase from Streptomyces coelicolor Has Two Alternative Catalytically Active Nucleophiles

Abstract

Fatty acids and polyketides are synthesized by mechanistically and evolutionarily related multienzyme systems. Their carbon chain backbones are synthesized via repeated decarboxylative condensations of alpha-carboxylated building blocks onto a growing acyl chain. These alpha-carboxylated building blocks are transferred from the corresponding coenzyme A thioesters onto the phosphopantetheine arm of an acyl carrier protein (ACP) by acyl transferases, which operate by a ping-pong mechanism involving an acyl-O-serine intermediate. 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 labeled by (14)C malonyl-CoA. Here we demonstrate that an alternative, catalytically competent nucleophile exists in the active site of this enzyme. Next to the active-site serine is a histidine residue that is conserved in some, 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 proteolysis, followed by radio-HPLC and mass spectrometric analysis of the S97A mutant enzyme. Kinetic analysis revealed that the k(cat) of the S97A mutant was within 10-fold that of the wild-type enzyme, whereas the K(M)s of the two enzymes were comparable. Sequence comparison with the E. coli MAT (whose X-ray structure is known) led to the identification of H201 as the putative base in the serine-histidine catalytic dyad of the S. coelicolor enzyme. The absence of MAT activity in the H201A mutant and the detection of weak activity in the H201Q mutant was consistent with this proposal. The implications of this unexpected finding are discussed.

Structure and Mechanism of the trans-Acting Acyltransferase from the Disorazole Synthase

“…Consistent with the conservation of the catalytic mechanism of all ATs, the active site of the DSZS AT is closely related in sequence and active site structure to other crystallographically characterized ATs [10, 11, 27]. The importance of a nucleophilic serine residue in the active site is supported by the behavior of the S86C mutant whose transacylation efficiency was reduced by 200-fold compared to the wild-type DSZS AT.…”

Crystal Structure of the Acyltransferase Domain of the Iterative Polyketide Synthase in Enediyne Biosynthesis

“…This mode of interaction, where the acetate moiety mimics interactions formed by the carboxylic end of the malonyl group, is similar to the ScMAT-acetate complex (38,43). However, the orientation of ACY1 in AT DYN differs slightly from the one adopted in ScMAT because ACY1 is positioned parallel to Ser 651 and almost perpendicular to the Arg 676 guanidino plane.…”

Fatty acid biosynthesis revisited: structure elucidation and metabolic engineering

“…88, 89 Interestingly, detailed mutagenesis of the active site residues of S. coelicolor MCAT suggests that only Ser97 is required for activity and not two implicated nucleophiles (Ser and His) as previously published. 90,91 …”