Stereospecificity Of Enoylreductase Domains From Modular Polyketide Synthases

Modular polyketide synthase(PKS)is a powerful biosynthetic factory that generates a large class of polyketides with complex structures and diverse activities.Biosynthesis of polyketides is formed via a Claisen-like decarboxylative condensation of short-chain carboxylic acids,which is similar with biosynthesis of fatty acids.However,PKSs utilize a wider range of both starter units and extension units,such as methyl malonyl CoAzyme A(CoA)to form a methyl substituent at C2 position of polyketide intermediate.Each PKSs module contains three functional domains necessary: an acyl transferase(AT),a ketosynthase(KS)and an acyl carrier protein(ACP).One or more modified domains are included,such as ketoreductase(KR),dehydratase(DH),and enoylreductase(ER).During the extension of polyketide,ER stereoselectively reduces the double bond generated by DH and controls the configuration of the C2-substituent.According to the stereoconfiguration of the C2 methyl group,ER is classified into S-type or R-type.To investigate the stereospecificity of ER,three S-type ERs were cloned and expressed.They were spinosyn PKS module 2(SpnER2),erythromycin PKS module 4(EryER4)and pikromycin PKS module 4(PikER4).The pantetheine(pant)-bound trans-2-methylcrotonyl substrate surrogates was reduced by ERs,and the products were mixture of 2S and 2R.The substrate surrogates of N-acetylcysteine(SNAC)-and coenzyme A(CoA)-were synthesized.Come as a surprise,they cannot be reduced by ERs.The natural intermediates are linked to the conserved serine residue of the acyl carrier protein(ACP)via a phosphopantetheine prosthetic group.To confirm the effect of ACP on ER stereospecificity,substrate analogs carried by SpnACP2,EryACP4,and PikACP4 were synthesized by phosphopantetheinyl transferases(sfp).When the cognate ACP-bound substrate surrogate was used,the expected 2S epimer was uniquely generated.The unexpected 2R epimer was increased in similar incubation of an ER with a noncognate ACP-bound substrate surrogate.These results highlighted the dependence of stereospecificity on proper protein-protein interactions between ER and ACP domains.Three conserved amino acids(tyrosine,lysine and aspartate)were detected by mutant assays.The results demonstrated the conserved tyrosine and lysine were involved in stereocontrol of ER.The rational design of PKS is an effective way to obtain new polyketides.These results extended our insights into the stereochemistry of ER and will contribute to the engineering of modular PKSs in the future.