HETEROLOGOUS EXPRESSION IN ESCHERICHIA-COLI OF AN INTACT MULTIENZYME COMPONENT OF THE ERYTHROMYCIN-PRODUCING POLYKETIDE SYNTHASE
HETEROLOGOUS EXPRESSION IN ESCHERICHIA-COLI OF AN INTACT MULTIENZYME COMPONENT OF THE ERYTHROMYCIN-PRODUCING POLYKETIDE SYNTHASE
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DOI：
10.1111/j.1432-1033.1993.tb17925.x
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发表时间：
1993-05-15
期刊：
影响因子：
--
通讯作者：
LEADLAY, PF
中科院分区：
文献类型：
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作者：
ROBERTS, GA;STAUNTON, J;LEADLAY, PF
6-脱氧红内酯B合成酶3(Debs 3)催化聚酮6-脱氧红内酯B的第五和第六个缩合循环，聚酮6-脱氧红内酯B是红霉生物合成红霉素A的第一个可分离中间体。此前，编码Debs 3的基因已经被克隆和测序，推导出的产物被预测在330 kDa的多肽链上包含9个脂肪酸合成酶样活性。该基因已被改造成基于PT-7的表达系统，在大肠杆菌中过度表达。诱导后的重组Debs-3蛋白占胞内可溶性蛋白的1-2%。从重组大肠杆菌抽提物中纯化的DeBS-3明显均一，未发现通过4‘-磷酸天冬氨酸的共价连接修饰。与聚酮链组装延伸单元的来源(R，S)-甲基丙二酰辅酶A孵育后，硫酯发生了水解，这意味着DeBS 3中的甲基丙二酰辅酶A：ACP酰基转移酶结构域被正确折叠并能够催化这一副反应。在这个反应中，Debs 3被瞬时标记，与酰基酶的中间体一致。凝胶过滤层析测得该蛋白的天然相对分子质量为668 kDa，对应于二聚体或高度不对称单体。
6-Deoxyerythronolide B synthase 3 (DEBS 3) is proposed to catalyse the fifth and sixth condensation cycles in the assembly of the polyketide 6-deoxyerythronolide B, the first isolatable intermediate in the biosynthesis of erythromycin A by Saccharopolyspora erythraea. The gene encoding DEBS 3 has previously been cloned and sequenced, and the deduced product is predicted to house nine fatty acid synthase-like activities on a 330-kDa polypeptide chain. The gene has been engineered into a pT-7-based expression system for over-expression in Escherichia coli. Recombinant DEBS 3 was found to constitute, after induction, 1-2% of soluble intracellular protein. DEBS 3 was purified from extracts of the recombinant E. coli to apparent homogeneity, and was found not to be modified by covalent attachment of the prosthetic group 4'-phosphopantetheine. Incubation with (R,S)-methylmalonyl-CoA, the presumed source of extension units for polyketide chain assembly, led to hydrolysis of the thioester, implying that the methylmalonyl-CoA:ACP acyltransferase domains in DEBS 3 are correctly folded and able to catalyse this side-reaction. During this reaction, DEBS 3 became transiently radiolabelled, consistent with the intermediacy of an acyl-enzyme. The native molecular mass of the protein by gel filtration chromatography was 668 kDa which corresponds either to a dimer or to a highly asymmetric monomer.