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 k(cat)/K(m) 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.

Figure 1. The Bacterial Fatty Acid Biosynthesis Pathway in E. coli

FabH (KASII), the β-ketoacyl-ACP synthase is responsible for condensing acetyl-CoA with malonyl-ACP to yield β-ketoacyl-ACP. This product is subsequently reduced, dehydrated and reduced by the successive action of a β-ketoacyl-ACP reductase, FabG, a dehydratase, FabZ or FabA, and a trans-2-enoyl-ACP reductase, FabI. A repetitive series of elongation reactions are then performed that utilize the same enzymes except that the condensation reaction is performed by the ACP specific β-ketoacyl synthases FabB (KASI) and FabF (KASII). In M. tuberculosis, FabH condenses malonyl-AcpM with long chain (C24+) acyl-CoAs that are provided by the mycobacterial FASI pathway. The homologues of the KASI, II and III enzymes in M. tuberculosis are KasA, KasB and mtFabH, respectively.

Figure 2. Structure of AcpM Colored to Show Peptides Derived from α-Helix 2

Structure of AcpM with the conserved serine on α-helix 2 colored in yellow. The lengths of the three synthesized peptide mimics are indicated in colors where the 8mer (DSLDMLEI-NH₂) is the yellow segment, the 14mer (DSLSMLEIAVQTED-NH₂) is the yellow plus the green segment and the 16mer (DPDSLSMLEIAVQTED-NH₂) is the yellow, green and red segments. The peptides were designed based on sequences of AcpM and ecACP together with information gleaned by Walsh et al who identified short peptides that could be phosphopantetheinylated by AcpS and Sfp (38). This figure was made using PyMol (52) and the PDB entry 1KLP (AcpM).

Figure 3. Structure of ecFabH-CoA Superimposed with ecFabF

The interaction of CoA with ecFabF was explored by superimposing the active sites and malonyl binding channels of each enzyme with the respective portions of ecFabH complexed with CoA (47, 52). (a) While R249 in ecFabH (green) forms an interaction to the pyrophosphate of the PPant chain, R226 in ecFabF (blue) is flipped 90 degrees and replaced by P308, preventing bond formation to the pyrophophate of CoA. (b) R151 in ecFabH which binds to the adenine portion of CoA is replaced by R206 in ecFabF which apparently cannot form the same favorable interaction. The figure was made using PyMol (52) and the PDB entries 2eft (ecFabH), 2gfw (ecFabF).

Figure 4. Proposed ecACP Binding Site on FabB

(a) The homodimeric structure of ecFabB (2VB9) in which monomer 1 is colored in purple and monomer 2 is colored in blue. The basic patch that forms the principal ACP binding site in monomer 1 is colored in yellow. R62, K63 and R66 are located on α-helix 3, while K151 is located on α-helix 9 all from monomer 1. Interaction of ACP with this basic patch would result in delivery of the substrate to monomer 2 and interaction of ACP with loop 1 (cyan) in monomer 2. (b) A detailed view of the locations of the conserved methionines that link the active site to helices α5 and α6 are shown in red. They are thought to alter their position upon ecACP binding and aid in the transition from free enzyme to the acyl-enzyme. M269 is located in the middle of loop 1 of monomer 2 and M137 is part of α-helix 6 of monomer 1. The figure was made using PyMOL (52).