Probing reactivity and substrate specificity of both subunits of the dimeric Mycobacterium tuberculosis FabH using alkyl-CoA disulfide inhibitors and acyl-CoA substrates.
The dimeric Mycobacterium tuberculosis FabH (mtFabH) catalyses a Claisen-type condensation between an acyl-CoA and malonyl-acyl carrier protein (ACP) to initiate the Type II fatty acid synthase cycle. To analyze the initial covalent acylation of mtFabH with acyl-CoA, we challenged it with mixture of C6-C20 acyl-CoAs and the ESI-MS analysis showed reaction at both subunits and a strict specificity for C12 acyl CoA. Crystallographic and ESI-MS studies of mtFabH with a decyl-CoA disulfide inhibitor revealed a decyl chain bound in acyl-binding channels of both subunits through disulfide linkage to the active site cysteine. These data provide the first unequivocal evidence that both subunits of mtFabH can react with substrates or inhibitor. The discrepancy between the observed C12 acyl-CoA substrate specificity in the initial acylation step and the higher catalytic efficiency of mtFabH for C18-C20 acyl-CoA substrates in the overall mtFabH catalyzed reaction suggests a role for M. tuberculosis ACP as a specificity determinant in this reaction.
Sachdeva
Sarbjot
S
Department of Chemistry, Portland State University, Portland, OR, USA.
Musayev
Faik
F
Alhamadsheh
Mamoun M
MM
Neel Scarsdale
J
J
Tonie Wright
H
H
Reynolds
Kevin A
KA
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000214:Acyl Coenzyme A; D000215:Acylation; D001426:Bacterial Proteins; D002384:Catalysis; D019281:Dimerization; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D021122:Protein Subunits; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D021122:Protein Subunits; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; C530127:FabH protein, Mycobacterium tuberculosis