The study of individual domains of rifamycin polyketide synthase provided detailed mechanical information of this megasynthase. This study was specially focused on the stereospecific reactivity of the dehydratase domain of rifamycin PKS module 10 (RifDH10) and the ketoreductase domain (RifKR10). Recombinant RifDH10 was coupled assayed with TylKR1 using in situ 2R-2-methyl-3-keto-DEBSACP6 which was generated by incubation of propionylSNAC with DEBS [KS6][AT6], DEBS ACP6 in the presence of methylmalonyl CoA and NADPH. The product was analyzed by GC/MS and radioactive TLC. The reaction produced in situ anti-(2R,3R)-2-methyl-3-hydroxyacyl-DEBS ACP6 which was further dehydrated by RifDH10, and the outcome was (E)-2-methylpent-2-enoyl-DEBS ACP6. RifDH10 was not active toward other diastereomers It was surprising that the enzyme did not process anti-(2S,3S)-methyl-hydroxyl-DEBS ACP6 considering that RifKR10-GST generated anti-(2S,3S)-methyl-hydroxyl-DEBS ACP6. In order to address this, a chemoenzymatically prepared acylated RifACP10 is being produced and tested with RifKR10-GST and RifDH10.  More detailed results will be presented.

Investigation of stereospecific reaction by the dehydratase domain and ketoreductase domain of rifamycin PKS module 10

244th National Meeting (2012)

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