Substrate recognition by β-ketoacyl-ACP synthases.
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.
Institute for Chemical Biology & Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, United States.
Janine G
JG
Borgaro
Andrew
A
Chang
Carl A
CA
Machutta
Xujie
X
Zhang
Peter J
PJ
Tonge
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003065:Coenzyme A / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054889:Fatty Acid Synthase, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010171:Palmitoyl Coenzyme A / Q000378:metabolism; D010204:Pantetheine / Q000031:analogs & derivatives / Q000737:chemistry; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D010171:Palmitoyl Coenzyme A; D010204:Pantetheine; D000123:Acetyltransferases; C557137:fabF protein, E coli; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; C490369:FabB protein, E coli; C530127:FabH protein, Mycobacterium tuberculosis; D017853:Phosphotransferases (Alcohol Group Acceptor); C023088:pantothenate kinase; D054889:Fatty Acid Synthase, Type II; C003129:4'-phosphopantetheine; D003065:Coenzyme A