Trapping Transient Complex Of Ketoreductase And Acyl Carrier Protein Domains Of Modular Polyketide Synthases By Covalently Cross-Linking

Modular polyketide synthases(mPKSs)are multifunctional enzyme assembly lines responsible for the biosynthesis of various polyketide with a diverse range of structures and activities.The acyl-carrier protein(ACP)domain serves as a middle tether for the precursors or intermediates in these pathways,collaborating with all catalytic enzyme partners.Despite the significance of these highly specific protein-protein interactions,elucidating the structures of the complex between the ACP domain and its partner domains remains challenging due to the instantaneous and reversible nature.Ketoreductase(KR)reduces the b-keto groups of the polyketide intermediates to D-or L-b-hydroxyl groups,and some of them can also isomerize the D-a-methyl groups to L-type ones.When lacking ACP(such as substrates derived from N-acetyl cysteamine(SNAC)),the product of KR's reduction using substrate analogs is usually a racemic mixture unsatisfactorily.It indicates the importance of specific KR-ACP interactions to the formation of the a-methyl and b-hydroxy chirality.Revealing these interactions could provide a foundation for directed modification of KRs to producing products with specific configurations.In order to capture a stable KR-ACP complex,the isolated KR and ACP domains were firstly covalently cross-linked by three bifunctional maleimide reagents,BMOE,BMB and BMH,possessing same reactivity but different spacer arm lengths.Since the weak binding affinity between ACP and KR,this simple cross-linking attempt didn't meet with success.The inadequate relative concentration may lead to a low probability of binding between KR and ACP and a difficulty for 4?-phosphopantetheine arm entering into the catalytic groove of KR in a free envirnment.Considering that in most modules of PKS,KR and ACP domains are fused together by a flexible peptide.The expression of Amp(KR+ACP)2fusion protein can increase the relative concentration of the two,which is conducive to the occurrence of further cross-linking.At the same time,inserting a tobacco etch virus(TEV)protease cleavage site in the peptide is facilitate to the confirmation of cross-linking.This di-domain fusion protein was incubated with cross-linkers,and the cross-linking was realized and later confirmed by TEV digestion.Afterwards,reaction conditions,including the incubation time,temperature,concentrations of protein and BMH,were optimized to increase the cross-linker efficiency.With different optimal cross-linking conditions,this strategy can be applied for KR and ACP domains from different modular PKSs,showing a certain degree of universality.According to the tags of proteins and their molecular weight,two different purifying methods suitable for “single label” and “double labels”fusion proteins were established respectively.If the fusion protein involved in the cross-linking reaction can be rightly expressed and purified with a single his-tag at the C-terminal,the “single label” purification method is suitable for further acquisition of the cross-linked complex.The generated complex was stable and pure for subsequent crystal structure analysis.But if the fusion protein could only be expressed and purified normally with a his-tag at the N-terminal,another small tag needs to be added at the C-terminal for subsequent acquisition of the cross-linked complex.Three small labels,Strep-,Flag-and HA-tag,have been tried and verified in this study.When the C-terminal of the fusion protein was attached with a Strep-tag or flag-tag,the final product contained a small amount of uncross-linked KR impurities.However,when attached with a HA-tag,the resulting product showed slight degradation.This study creatively developed and verified the di-domain covalent cross-linking strategy,using which the transient complex of ketoreductase and homologous acyl carrier protein was successfully captured.Purification methods were established to obtain pure KR-ACP complex.This study provided experimental basis for the later KR-ACP complex crystal structure analysis,the interaction mechanism study between KR and ACP,and the identification of key amino acids involved.It's hoped that this study will facilitate the rational design of stereospecific KR and obtain more desirable new polyketides.