Functional reconstitution of the Mycobacterium tuberculosis long-chain acyl-CoA carboxylase from multiple acyl-CoA subunits.
Functional reconstitution of the Mycobacterium tuberculosis long-chain acyl-CoA carboxylase from multiple acyl-CoA subunits.
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DOI：
10.1111/febs.14046
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发表时间：
2017-04
期刊：
影响因子：
--
通讯作者：
Gramajo H
中科院分区：
文献类型：
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作者：
Bazet Lyonnet B;Diacovich L;Gago G;Spina L;Bardou F;Lemassu A;Quémard A;Gramajo H
结核分枝杆菌产生大量结构多样的脂质，这些脂质与该生物体的致病性、持久性和抗生素抗性有关。参与所有这些脂质的生物合成的大多数构件由酰基辅酶A羧化酶（ACCase）产生，其亚基组成和生理作用尚未明确确定。文献中一个相当有争议的数据涉及产生长链α-羧基酰基-CoA的酶复合物的确切蛋白质组成和底物特异性;该酶复合物是参与聚酮合酶Pks 13介导的缩合最后一步以合成成熟分枝菌酸的底物之一。我们成功地从纯化的长链酰基辅酶A羧化酶复合物（LCC）的α-亚基AccA 3、ε-亚基AccE 5和两个β-亚基AccD 4和AccD 5中重组了LCC，并证明这四个亚基是LCC活性所必需的。此外，我们还通过底物竞争实验和AccD 5亚基的特异性抑制剂的使用表明，其在长酰基辅酶A的羧化中的作用，作为LCC复合物的一部分，是结构性的而不是催化性的。此外，在LCC酶复合物的背景下，AccD 5还能够羧酸化其天然底物乙酰辅酶A和丙酰辅酶A。因此，由这四个亚基形成的超复合物具有产生主要底物丙二酰辅酶A、甲基丙二酰辅酶A和α-羧基-C24 -26-辅酶A的潜力，用作该病原体中存在的所有脂质的生物合成的缩合单元。一种新的长链酰基辅酶A羧化酶复合物（LCC）由一个生物素化的α亚基（AccA 3）、两个不同的羧基转移酶亚基β（AccD 4）和β′（AccD 5）以及一个ε亚基（AccE 5）形成，其特征在于能够羧化短链和长链酰基辅酶A。该超复合物可为脂肪酸和分枝菌酸的生物合成以及甲基化脂质的生物合成提供底物。结核
Mycobacterium tuberculosis produces a large number of structurally diverse lipids that have been implicated in the pathogenicity, persistence and antibiotic resistance of this organism. Most building blocks involved in the biosynthesis of all these lipids are generated by acyl-CoA carboxylases (ACCase) whose subunit composition and physiological roles have not yet been clearly established. A rather controversial data in the literature refers to the exact protein composition and substrate specificity of the enzyme complex that produces the long-chain α-carboxy-acyl-CoAs; one of the substrates involved in the last step of condensation mediated by the polyketide synthase Pks13 to synthesize mature mycolic acids. Here we have successfully reconstituted the so called long-chain acyl-CoA carboxylase complex (LCC) from its purified components: the α-subunit AccA3, the ε-subunit AccE5 and the two β-subunits AccD4 and AccD5, and demonstrated that the four subunits are essential for its LCC activity. Furthermore, we also showed by substrate competition experiments and the use of a specific inhibitor of the AccD5 subunit, that its role in the carboxylation of the long acyl-CoAs, as part of the LCC complex, was structural rather than catalytic. Moreover, AccD5 was also able to carboxylate its natural substrates, acetyl-CoA and propionyl-CoA, in the context of the LCC enzyme complex. Thus, the supercomplex formed by these four subunits has the potential to generate the main substrates, malonyl-CoA, methylmalonyl-CoA and α-carboxy-C24–26-CoA, used as condensing units for the biosynthesis of all the lipids present in this pathogen. A new long-chain acyl-CoA carboxylase complex (LCC) formed by a biotinylated α subunit (AccA3), two different carboxyltransferase subunits β(AccD4) and β′ (AccD5) and an ε subunit (AccE5), was characterized and showed to be able to carboxylate short- as well as long-chain acyl-CoAs. This supercomplex could provide the substrates needed for fatty acids and mycolic acid biosynthesis, as well as for methyl-branched lipid biosynthesis of M. tuberculosis.