ja408944s_si_001.pdf (1.77 MB)
Coupled Methyl Group Epimerization and Reduction by Polyketide Synthase Ketoreductase Domains. Ketoreductase-Catalyzed Equilibrium Isotope Exchange
journal contribution
posted on 2013-11-06, 00:00 authored by Ashish Garg, Chaitan Khosla, David E. CaneIncubation
of [2-2H]-(2S,3R)-2-methyl-3-hydroxypentanoyl-SACP
([2-2H]-1a) with the epimerizing ketoreductase
domain EryKR1
in the presence of a catalytic amount NADP+ (0.05 equiv)
resulted in time- and cofactor-dependent washout of deuterium from 1a, as a result of equilibrium isotope exchange of transiently
generated [2-2H]-2-methyl-3-ketopentanoyl-ACP. Incubations
of [2-2H]-(2S,3S)-2-methyl-3-hydroxy-pentanoyl-SACP
with RifKR7 and with NysKR1 also resulted in time-dependent loss of
deuterium. By contrast, incubations of [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP
and [2-2H]-(2R,3R)-2-methyl-3-hydroxypentanoyl-SACP
with the non-epimerizing ketoreductase domains EryKR6 and TylKR1,
respectively, did not result in any significant washout of deuterium.
The isotope exchange assay directly establishes that specific polyketide
synthase ketoreductase domains also have an intrinsic epimerase
activity, thus enabling mechanistic analysis of a key determinant
of polyketide stereocomplexity.