title: Biochemical analysis of the substrate specificity of the beta-ketoacyl-acyl carrier protein synthase domain of module 2 of the erythromycin polyketide synthase.
abstract: The beta-ketoacyl-acyl carrier protein synthase (KS) domain of the modular 6-deoxyerythronolide B synthase (DEBS) catalyzes the fundamental chain building reaction of polyketide biosynthesis. The KS-catalyzed reaction involves two discrete steps consisting of formation of an acyl-enzyme intermediate generated from the incoming acylthioester substrate and an active site cysteine residue, and the conversion of this intermediate to the beta-ketoacyl-acyl carrier protein product by a decarboxylative condensation with a paired methylmalonyl-SACP. We have determined the rate constants for the individual biochemical steps by a combination of protein acylation and transthioesterification experiments. The first-order rate constant (k(2)) for formation of the acyl-enzyme intermediate from [1-(14)C]-(2S,3R)-2-methyl-3-hydroxypentanoyl-SNAC (2) and recombinant DEBS module 2 is 5.8 +/- 2.6 min(-)(1), with a dissociation constant (K(S)) of 3.5 +/- 2.8 mM. The acyl-enzyme adduct was formed at a near-stoichiometric ratio of approximately 0.8:1. Transthioesterification between unlabeled diketide-SNAC 2 and N-[1-(14)C-acetyl]cysteamine gave a k(exch) of 0.15 +/- 0.06 min(-)(1), with a K(m) for HSNAC of 5.7 +/- 4.9 mM and a K(m) for 2 of 5.3 +/- 0.9 mM. Under the conditions that were used, k(exch) was equal to k(-)(2), the first-order rate constant for reversal of the acyl-enzyme-forming reaction. Since the rate of the decarboxylative condensation is much greater that the rate of reversion to the starting material (k(3) >> k(-)(2)), formation of the acyl-enzyme adduct is effectively irreversible, thereby establishing that the observed value of the specificity constant (k(cat)/K(m)) is solely a reflection of the intrinsic substrate specificity of the KS-catalyzed acyl-enzyme-forming reaction. These findings were also extended to a panel of diketide- and triketide-SNAC analogues, revealing that some substrate analogues that are not converted to product by DEBS module 2 form dead-end acyl-enzyme intermediates.
authors.affiliation: Department of Chemistry, Box H, Brown University, Providence, Rhode Island 02912-9108, USA.
authors.forename: Jiaquan
authors.initials: J
authors.lastname: Wu
authors.forename: Kenji
authors.initials: K
authors.lastname: Kinoshita
authors.forename: Chaitan
authors.initials: C
authors.lastname: Khosla
authors.forename: David E
authors.initials: DE
authors.lastname: Cane
mesh_terms: D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000235:genetics / Q000378:metabolism; D000215:Acylation; D002250:Carbon Radioisotopes / Q000378:metabolism; D007700:Kinetics; D048630:Polyketide Synthases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D002250:Carbon Radioisotopes; D048630:Polyketide Synthases; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase