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硫辛酸合成:一类新的辛酰基转移酶家族,通常被注释为脂酰基辅酶 A 连接酶。

Lipoic acid synthesis: a new family of octanoyltransferases generally annotated as lipoate protein ligases.

机构信息

Department of Microbiology, University of Illinois, Urbana, Illinois 61801, United States.

出版信息

Biochemistry. 2010 Nov 23;49(46):10024-36. doi: 10.1021/bi101215f. Epub 2010 Oct 27.

Abstract

Bacillus subtilis lacks a recognizable homologue of the LipB octanoyltransferase, an enzyme essential for lipoic acid synthesis in Escherichia coli. LipB transfers the octanoyl moiety from octanoyl-acyl carrier protein to the lipoyl domains of the 2-oxoacid dehydrogenases via a thioester-linked octanoyl-LipB intermediate. The octanoylated dehydrogenase is then converted to the enzymatically active lipoylated species by insertion of two sulfur atoms into the octanoyl moiety by the S-adenosyl-l-methionine radical enzyme, LipA (lipoate synthase). B. subtilis synthesizes lipoic acid and contains a LipA homologue that is fully functional in E. coli. Therefore, the lack of a LipB homologue presented the puzzle of how B. subtilis synthesizes the LipA substrate. We report that B. subtilis encodes an octanoyltransferase that has virtually no sequence resemblance to E. coli LipB but instead has a sequence that resembles that of the E. coli lipoate ligase, LplA. On the basis of this resemblance, these genes have generally been annotated as encoding a lipoate ligase, an enzyme that in E. coli scavenges lipoic acid from the environment but plays no role in de novo synthesis. We have named the B. subtilis octanoyltransferase LipM and find that, like LipB, the LipM reaction proceeds through a thioester-linked acyl enzyme intermediate. The LipM active site nucleophile was identified as C150 by the finding that this thiol becomes modified when LipM is expressed in E. coli. The level of the octanoyl-LipM intermediate can be significantly decreased by blocking fatty acid synthesis during LipM expression, and C150 was confirmed as an essential active site residue by site-directed mutagenesis. LipM homologues seem the sole type of octanoyltransferase present in the firmicutes and are also present in the cyanobacteria. LipM type octanoyltransferases represent a new clade of the PF03099 protein family, suggesting that octanoyl transfer activity has evolved at least twice within this superfamily.

摘要

枯草芽孢杆菌缺乏可识别的同源物 LipB 辛酰基转移酶,该酶是大肠杆菌中脂酰基辅酶 A 合成所必需的。LipB 通过硫酯连接的辛酰基-LipB 中间产物将辛酰基部分从辛酰基酰基载体蛋白转移到 2-氧代酸脱氢酶的脂酰基结构域。然后,通过 S-腺苷甲硫氨酸自由基酶 LipA(脂酰基辅酶 A 合成酶)将两个硫原子插入辛酰基部分,将辛酰化的脱氢酶转化为具有酶活性的脂酰化形式。枯草芽孢杆菌合成脂酰基辅酶 A,并且含有在大肠杆菌中完全功能的 LipA 同源物。因此,缺乏 LipB 同源物提出了枯草芽孢杆菌如何合成 LipA 底物的难题。我们报告说,枯草芽孢杆菌编码一种辛酰基转移酶,该酶与大肠杆菌 LipB 几乎没有序列相似性,但序列与大肠杆菌脂酰基辅酶 A 连接酶 LplA 相似。基于这种相似性,这些基因通常被注释为编码脂酰基辅酶 A 连接酶,该酶在大肠杆菌中从环境中摄取脂酰基辅酶 A,但在从头合成中不起作用。我们将枯草芽孢杆菌的辛酰基转移酶命名为 LipM,并发现,与 LipB 一样,LipM 反应通过硫酯连接的酰基酶中间产物进行。通过发现当 LipM 在大肠杆菌中表达时,该硫醇会被修饰,从而确定了 LipM 的活性位点亲核试剂为 C150。通过在 LipM 表达期间阻断脂肪酸合成,可以显著降低辛酰基-LipM 中间产物的水平,并且通过定点突变确认 C150 是必需的活性位点残基。LipM 同源物似乎是厚壁菌门中唯一类型的辛酰基转移酶,也存在于蓝细菌中。LipM 型辛酰基转移酶代表 PF03099 蛋白家族的一个新分支,表明辛酰基转移活性在这个超家族中至少进化了两次。

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