pH-Rate profiles establish that polyketide synthase dehydratase domains utilize a single-base mechanism.

FosDH1 from module 1 of the fostriecin polyketide synthase (PKS) catalyzes the dehydration of a 3-hydroxybutyryl-SACP to the (E)-3-butenoyl-SACP. The steady-state kinetic parameters, kcat and kcat/Km, were determined over the pH range 3.0 to 9.2 for the FosDH1-catalyzed dehydration of the N-acetycsteamine thioester, 3-hydroxybutyryl-SNAC (3), to (E)-3-butenoyl-SNAC (4). The pH rate profiles for both log(kcat) and log(kcat/Km) each corresponded to a single pH-dependent ionization to give an active site general base, with a calculated pKa 6.1 ± 0.2 for kcat and pKa 5.7 ± 0.1 for kcat/Km. These results are inconsistent with the commonly suggested "two-base" (base-acid) mechanism for the dehydratases of PKS and fatty acid biosynthesis and support a simple one-base mechanism in which the universally conserved active site His residue acts as the base to deprotonate C-2 of the substrate, then redonates the proton to the C-3 hydroxyl group to promote C-O bond-cleavage and elimination of water. The carboxylate of the paired Asp or Glu residue is thought to bind and orient the hydroxyl group of the substrate in the stereoelectonically favored conformation.