Biochemical Analysis of the Substrate Specificity of the β-Ketoacyl-Acyl Carrier Protein Synthase Domain of Module 2 of the Erythromycin Polyketide Synthase

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Abstract
The β-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 β-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 (k2) for formation of the acyl−enzyme intermediate from [1-14C]-(2S,3R)-2-methyl-3-hydroxypentanoyl-SNAC (2) and recombinant DEBS module 2 is 5.8 ± 2.6 min-1, with a dissociation constant (KS) of 3.5 ± 2.8 mM. The acyl−enzyme adduct was formed at a near-stoichiometric ratio of ∼0.8:1. Transthioesterification between unlabeled diketide-SNAC 2 and N-[1-14C-acetyl]cysteamine gave a kexch of 0.15 ± 0.06 min-1, with a Km for HSNAC of 5.7 ± 4.9 mM and a Km for 2 of 5.3 ± 0.9 mM. Under the conditions that were used, kexch 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 (k3 ≫ k-2), formation of the acyl−enzyme adduct is effectively irreversible, thereby establishing that the observed value of the specificity constant (kcat/Km) 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.