Substrate Recognition by β-Ketoacyl-ACP Synthases

Abstract

β-Ketoacyl-ACP synthase (KAS) enzymes catalyze Claisen condensation reactions in the fatty acid biosynthesis pathway. These reactions follow a ping-pong mechanism in which a donor substrate acylates the active site cysteine residue after which the acyl group is condensed with the malonyl-ACP acceptor substrate to form a β-ketoacyl-ACP. In the priming KASIII enzymes the donor substrate is an acyl-CoA while in the elongating KASI and KASII enzymes the donor is an acyl-ACP. Although the KASIII enzyme in Escherichia coli (ecFabH) is essential, the corresponding enzyme in Mycobacterium tuberculosis (mtFabH) is not, suggesting that the KASI or II enzyme in M. tuberculosis (KasA or KasB, respectively) must be able to accept a CoA donor substrate. Since KasA is essential, the substrate specificity of this KASI enzyme has been explored using substrates based on phosphopantetheine, CoA, ACP and AcpM peptide mimics. This analysis has been extended to the KASI and KASII enzymes from E. coli (ecFabB and ecFabF) where we show that a 14 residue malonyl-phosphopantetheine peptide can efficiently replace malonyl-ecACP as the acceptor substrate in the ecFabF reaction. While ecFabF is able to catalyze the condensation reaction when CoA is the carrier for both substrates, the KASI enzymes ecFabB and KasA have an absolute requirement for an ACP substrate as the acyl donor. Provided that this requirement is met, variation in the acceptor carrier substrate has little impact on the kcat/Km for the KASI reaction. For the KASI enzymes we propose that the binding of ecACP (AcpM) results in a conformational change that leads to an open form of the enzyme to which the malonyl acceptor substrate binds. Finally, the substrate inhibition observed when palmitoyl-CoA is the donor substrate for the KasA reaction has implications for the importance of mtFabH in the mycobacterial FASII pathway.

Structural Basis for the Recognition of Mycolic Acid Precursors by KasA, a Condensing Enzyme and Drug Target from Mycobacterium Tuberculosis

“…D, proposed AcpM binding site. Basic residues located on ␣3Ј and 3Ј probably bind to the acidic AcpM protein, which is believed to deliver its substrate to the adjacent subunit (45). A HEPES buffer molecule (shown in cyan) bound to the ␣3Ј basic patch was observed for the C171Q KasA and C171Q KasA-TLM structures.…”

“…In support of this hypothesis, we observe that the acyl chain itself is not able to induce these conformational changes, as exemplified by the absence of the phospholipids in the wild-type KasA binding cavity. Furthermore, our model explains why acyl-CoA substrates are not efficiently processed by KasA (45). In contrast, the FabH enzyme harbors a fundamentally different capping region (supplemental Fig.…”

“…In a recent kinetic study, Borgaro et al (45) proposed that the binding of the acidic ACP protein to one ecFabB monomer leads to the delivery of its substrate to the active site of the adjacent subunit. They identified several basic amino acids on the protein surface that are important for ACP binding.…”

“…4D) (45), and thus it is likely that AcpM binding initiates this process. Based on this hypothesis, we propose the following mechanism.…”

Bacterial fatty acid metabolism in modern antibiotic discovery

“…FabB has similar function as FabF, but is essential for the elongation of unsaturated fatty acids in bacteria expressing a FabA [49,50]. FabF and FabB have a Cys-His-His catalytic triad, and FabH has a Cys-His-Asn triad [51]. Condensing enzymes proceed via a Ping-Pong mechanism where the active site cysteine attacks the thioester of the acetyl-CoA to make the acyl-cysteine thioester intermediate [52-54].…”