Zhao Xin, Miller J Richard, Cronan John E
Department of Microbiology, University of Illinois, Urbana, Illinois 61801, USA.
Biochemistry. 2005 Dec 20;44(50):16737-46. doi: 10.1021/bi051865y.
The lipB gene of Escherichia coli encodes an enzyme (LipB) that transfers the octanoyl moiety of octanoyl-acyl carrier protein (octanoyl-ACP) to the lipoyl domains of the 2-oxo acid dehydrogenases and the H subunit of glycine cleavage enzyme. We report that the LipB reaction proceeds through an acyl-enzyme intermediate in which the octanoyl moiety forms a thioester bond with the thiol of residue C169. The intermediate was catalytically competent in that the octanoyl group of the purified octanoylated LipB was transferred either to an 87-residue lipoyl domain derived from E. coli pyruvate dehydrogenase or to ACP (in the reversal of the physiological reaction). The octanoylated LipB linkage was cleaved by thiol reagents and by neutral hydroxylamine, strongly suggesting a thioester bond. Separation and mass spectral analyses of the peptides of the unmodified and octanoylated proteins showed that each of the assigned peptides of the two proteins had identical masses, indicating that none of these peptides were octanoylated. However, the one major peptide that we failed to recover was that predicted to contain all three LipB cysteine residues. These three cysteine residues were therefore targeted for site-directed mutagenesis and only C169 was found to be essential for LipB function in vivo. The C169S protein had no detectable activity whereas the C169A protein retained trace activity. Surprisingly, both proteins lacking C169 formed an octanoyl-LipB species, although neither was catalytically competent. The octanoyl-LipB species formed by the C169S protein was resistant to neutral hydroxylamine treatment, consistent with formation of an ester linkage to the serine hydroxyl group. The octanoyl-C169A LipB species was probably acylated at C147. LipB species that lacked all three cysteine residues also formed a catalytically incompetent octanoyl adduct, indicating the presence of a reactive side chain other than a cysteine thiol that lies adjacent to the active site.
大肠杆菌的lipB基因编码一种酶（LipB），该酶可将辛酰基 - 酰基载体蛋白（辛酰基 - ACP）的辛酰基部分转移至2 - 氧代酸脱氢酶的硫辛酰结构域以及甘氨酸裂解酶的H亚基。我们报告称，LipB反应通过一种酰基 - 酶中间体进行，其中辛酰基部分与残基C169的硫醇形成硫酯键。该中间体具有催化活性，因为纯化的辛酰化LipB的辛酰基可转移至源自大肠杆菌丙酮酸脱氢酶的87个残基的硫辛酰结构域或ACP（生理反应的逆反应）。辛酰化LipB的连接可被硫醇试剂和中性羟胺裂解，强烈表明存在硫酯键。未修饰和辛酰化蛋白的肽段的分离和质谱分析表明，这两种蛋白的每个指定肽段质量相同，表明这些肽段均未被辛酰化。然而，我们未能回收的一个主要肽段是预计包含LipB所有三个半胱氨酸残基的肽段。因此，针对这三个半胱氨酸残基进行了定点诱变，结果发现只有C169对于LipB在体内的功能至关重要。C169S蛋白没有可检测到的活性，而C169A蛋白保留了微量活性。令人惊讶的是，两种缺乏C169的蛋白都形成了一种辛酰基 - LipB物种，尽管两者都没有催化活性。由C169S蛋白形成的辛酰基 - LipB物种对中性羟胺处理具有抗性，这与形成与丝氨酸羟基的酯键一致。辛酰基 - C169A LipB物种可能在C147处被酰化。缺乏所有三个半胱氨酸残基的LipB物种也形成了一种无催化活性的辛酰加合物，表明除了与活性位点相邻的半胱氨酸硫醇之外，还存在一个反应性侧链。
