Essential Role of the
Donor Acyl Carrier Protein in
Stereoselective Chain Translocation to a Fully Reducing Module of
the Nanchangmycin Polyketide Synthase
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Abstract
Incubation of recombinant module 2 of the polyether nanchangmycin
synthase (NANS), carrying an appended thioesterase domain, with the
ACP-bound substrate (2<i>RS</i>)-2-methyl-3-ketobutyryl-NANS_ACP1
(<b>2-ACP1</b>) and methylmalonyl-CoA in the presence of NADPH
gave diastereomerically pure (2<i>S</i>,4<i>R</i>)-2,4-dimethyl-5-ketohexanoic acid (<b>4a</b>). These results
contrast with the previously reported weak discrimination by NANS
module 2+TE between the enantiomers of the corresponding <i>N</i>-acetylcysteamine-conjugated substrate analogue (±)-2-methyl-3-ketobutyryl-SNAC
(<b>2-SNAC</b>), which resulted in formation of a 5:3 mixture
of <b>4a</b> and its (2<i>S</i>,4<i>S</i>)-diastereomer <b>4b</b>. Incubation of NANS module 2+TE with <b>2-ACP1</b> in the absence of NADPH gave unreduced 3,5,6-trimethyl-4-hydroxypyrone
(<b>3</b>) with a <i>k</i><sub>cat</sub> of 4.4 ±
0.9 min<sup>–1</sup> and a <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> of 67 min<sup>–1</sup> mM<sup>–1</sup>, corresponding to a ∼2300-fold increase compared
to the <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> for the diffusive substrate <b>2-SNAC</b>. Covalent tethering
of the 2-methyl-3-ketobutyryl thioester substrate to the NANS ACP1
domain derived from the natural upstream PKS module of the nanchangmycin
synthase significantly enhanced both the stereospecificity and the
kinetic efficiency of the sequential polyketide chain translocation
and condensation reactions catalyzed by the ketosynthase domain of
NANS module 2