The loading and initial elongation modules of rifamycin synthetase collaborate to produce mixed aryl ketide products.
Rifamycin synthetase assembles the chemical backbone that members of the rifamycin family of antibiotics have in common. The synthetase contains a mixed biosynthetic interface between its loading module, which uses a nonribosomal peptide synthetase mechanism, and its initial elongation module, which uses a polyketide synthase mechanism. Biochemical studies of the loading and initial elongation modules of rifamycin synthetase reveal that this bimodular protein (LM-M1) catalyzes the formation of the phenyl ketide 3-hydroxy-2-methyl-3-phenylpropionate via a series of reactions that require benzoate, Mg.ATP, methylmalonyl-CoA, and NADPH. The overall rate of phenyl ketide production appears to be determined by the covalent loading of benzoate onto LM-M1, rather than by subsequent steps such as intermodular transfer of benzoate or condensation of benzoate and methylmalonate. Substituted benzoates that have previously been shown to be substrates for the loading module alone can also be incorporated into the corresponding aryl ketides by LM-M1, suggesting that the bimodular protein has a broad substrate tolerance. Discrimination between the substituted benzoates appears to reside in the benzoate loading reaction, and preincubation of LM-M1 with substituted benzoates and Mg.ATP allows faster downstream reactions to be unmasked. LM-M1 may be a useful biochemical system for exploring interactions between nonribosomal peptide synthetase and polyketide synthase modules.
Admiraal
Suzanne J
SJ
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Khosla
Chaitan
C
Walsh
Christopher T
CT
D000215:Acylation; D062365:Aminobenzoates / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D062385:Hydroxybenzoates; D007700:Kinetics; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010441:Peptide Chain Elongation, Translational; D010453:Peptide Synthases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010666:Phenylpropionates / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism; D012294:Rifamycins / Q000096:biosynthesis*; D013379:Substrate Specificity
D062365:Aminobenzoates; D000900:Anti-Bacterial Agents; D062385:Hydroxybenzoates; D009097:Multienzyme Complexes; D010666:Phenylpropionates; D011993:Recombinant Fusion Proteins; D012294:Rifamycins; C035253:3-phenylpropionic acid; C031509:3-amino-5-hydroxybenzoic acid; D010453:Peptide Synthases