Structural And Functional Analysis Of Ketoreductase Domains From Modular Polyketide Synthases

Polyketides are a kind of secondary metabolites that are synthetised by Polyketide synthases(PKSs),with having numerous chiral centers as one of theirt significant structural characteristic.During the process of their synthesis,ketoreductase(KR)domains of modular polyketide synthases control the the stereochemistry of C2 methyl and C3 hydroxyl substituents of polyketide intermediates.A-type KRs yeild products of L-hydroxyl,whereas B-type KRs yeild D-hydroxyl groups.The conserved“W”(in A-type)and LDD(in B-type)sequencemotifs reside on opposite sides of the catalytic grooves.Here we cloned a KR from the second module of the amphotericin PKS(AmpKR2)and the crystal structure of the KR complexed with NADP~+and 2-methyl-3-oxopentanoyl-pantetheine(MOPP)was solved.The structure shows that the conserved“W”of the KR forms hydrogen bonds with pantetheine handle of thesubstrate,guiding MOPP enters into the active site of an A-type KR from the side opposite the coenzyme to expose the re-face of C3 ketone to the 4'-pro-S-hydride to yeild an L-hydroxyl substituent.AmpKR2 which preserves the D-orientation of the C2-methyl group is classified as A1-type.But there are some other KRs which have a conserved“H”in their active centers can catalyze the epimerization of D-methyl to yeild L-methyl.They are called A2-type KRs.The G355T/Q364H mutant of AmpKR2 works as an A2-type KR when catalysising the reduction of 2-methyl-3-oxopentanoyl-N-acetyl cysteamine(MOPN).We acquired crystal structure of ternary complex of AmpKR2G355T/Q364HmutantwithNADP~+and2-methyl-3-oxopentanoyl-pantetheine.Comparing with the structure of wild-type AmpKR2,the 2-methyl-3-oxopentanoyl group binding pocket of the mutant is a little bigger.Steady-state kinetic parameters of AmpKR2shows that Km towards MOPP is apparently smaller than that towards MOPN,which is consistent with the fact that we can only see MOPP but MOPN in the active center in the crystal structure.Functional assays show that AmpKR2 produces only A1(2D,3L)type intermediates as expected,while AmpKR2 G355T/Q364H produces both A1 type(~57%)and A2type(2L,3L,~43%)products at the same time.which indicates that the pantetheine handle has a a significant effect on stereospecificity of the KRs.Together,these findings extend our current understanding of the stereochemical control of PKS KR domains,providing basis to the design and modification of KRs in a certain extent.