垩唑霉素生物合成的反式酰基转移酶具有底物宽泛性

Trans-acyltransferase of Oxazolomycin Biosynthesis Has Substrate Broadness

垩唑霉素生物合成中的聚酮合酶(PKS)均缺少酰基转移酶(AT)功能域,在聚酮合酶外含有两个独立的反式AT OzmM和OzmC。对反式AT的敲除及回补实验证明两个反式AT是垩唑霉素合成所必需的。AT的功能是将延伸单元丙二酰辅酶A或者甲基丙二酰辅酶A传递到酰基载体蛋白(ACP)。为了研究OzmM和OzmC在垩唑霉素生物合成中的功能,本研究以OzmM蛋白和PKS蛋白OzmK为研究对象,研究了反式AT是否和PKS蛋白存在相互作用及反式AT将延伸单元传递给ACP后是否仍和PKS蛋白存在相互作用。为确定OzmM的功能,本研究在大肠杆菌BL21(DE3)中表达了OzmM蛋白并对其纯化进行体外实验,并且利用亲和共纯化和生物膜干涉技术验证了OzmM和OzmK之间的相互作用。本研究推测当OzmM将底物传递给ACP后会离开PKS蛋白,不参与延伸单元与聚酮主链的缩合反应,并且反式AT(OzmM)与PKS(OzmK)之间的弱相互作用是反式AT在ACP与PKS之间快速传递延伸单元的功能所必须的。另一个反式AT OzmC的功能为传递甲氧丙二酰ACP到OzmJ-ACP,本研究利用丙二酰辅酶A、甲基丙二酰辅酶A对其底物宽泛性进行研究,发现OzmC可以将丙二酰辅酶A传递给OzmQ-ACP,但不可以传递甲基丙二酰辅酶A。

The polyketide synthases involved in the biosynthesis of oxazolomycin lack the acyltransferase(AT)domain and there are two trans acyltransferases,OzmM and OzmC outside the PKS.Knockout and replenishment experiments with trans-AT gene demonstrated that two trans-ATs are required for the synthesis of oxazolomycin.The function of the ATs is to deliver the extension unit malonyl-CoA or methylmalonyl-CoA to the acyl carrier protein(ACP).The OzmM protein and PKS protein OzmK are the research object to study whether trans AT interacts with PKS protein and whether trans AT still interacts with PKS protein after delivery.To identify the function of OzmM,we expressed OzmM protein in E.coli BL21(DE3),and purified it to do the in vitro experiment Then we used the method of affinity co-purification and biolayer interferometry to study the interaction of OzmM and OzmK protein.We speculate that when OzmM transfers a substrate to ACP it leaves the PKS protein and does not participate in the next extension of the polyketide backbone.Moreover the weak interaction between trans AT(OzmM)and PKS(OzmK)is necessary for this function of trans AT to rapidly transfer the extension unit between ACP and PKS.Another trans AT OzmC was responsible for delivering methoxymalonyl-ACP to OzmJ-ACP.We used malonyl-CoA and methylmalonyl-CoA to study the tolerance of the substrate of OzmC and found that OzmC could transfer malonyl-CoA to OzmQ-ACP but it could not transfer methylmalonyl-CoA to ACP.