Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada T2N 4N1.
Biochemistry. 2010 Jun 8;49(22):4687-94. doi: 10.1021/bi901612y.
Holocarboxylase synthetase (HCS, human) and BirA (Escherichia coli) are biotin protein ligases that catalyze the ATP-dependent attachment of biotin to apocarboxylases. Biotin attachment occurs on a highly conserved lysine residue within a consensus sequence (Ala/Val-Met-Lys-Met) that is found in carboxylases in most organisms. Numerous studies have indicated that HCS and BirA, as well as biotin protein ligases from other organisms, can attach biotin to apocarboxylases from different organisms, indicating that the mechanism of biotin attachment is well conserved. In this study, we examined the cross-reactivity of biotin attachment between human and bacterial biotin ligases by comparing biotinylation of p-67 and BCCP87, the biotin-attachment domain fragments from human propionyl-CoA carboxylase and E. coli acetyl-CoA carboxylase, respectively. While BirA has similar biotinylation activity toward the two substrates, HCS has reduced activity toward bacterial BCCP87 relative to its native substrate, p-67. The crystal structure of a digested form of p-67, spanning a sequence that contains a seven-residue protruding thumb loop in BCCP87, revealed the absence of a similar structure in the human peptide. Significantly, an engineered "thumbless" bacterial BCCP87 could be biotinylated by HCS, with substrate affinity restored to near normal. This study suggests that the thumb loop found in bacterial carboxylases interferes with optimal interaction with the mammalian biotin protein ligase. While the function of the thumb loop remains unknown, these results indicate a constraint on specificity of the bacterial substrate for biotin attachment that is not itself a feature of BirA.
羟基亚戊二酰基辅酶 A 合酶（HCS，人源）和 BirA（大肠杆菌）是生物素蛋白连接酶，能够催化生物素与脱羧酶的依赖于 ATP 的连接。生物素的附着发生在羧化酶中高度保守的赖氨酸残基上，该赖氨酸残基位于大多数生物中存在的共识序列（Ala/Val-Met-Lys-Met）内。大量研究表明，HCS 和 BirA 以及其他生物体的生物素蛋白连接酶可以将生物素附着到来自不同生物体的脱羧酶上，这表明生物素附着的机制得到了很好的保守。在这项研究中，我们通过比较人源丙酰基辅酶 A 羧化酶的生物素结合域片段 p-67 和大肠杆菌乙酰辅酶 A 羧化酶的生物素结合域片段 BCCP87 的生物素化，来研究人源和细菌生物素连接酶之间的生物素结合的交叉反应性。虽然 BirA 对这两种底物具有相似的生物素化活性，但 HCS 对细菌 BCCP87 的活性相对于其天然底物 p-67 降低。p-67 消化形式的晶体结构，跨越了 BCCP87 中存在一个七残基突出的拇指环的序列，揭示了人源肽中不存在类似结构。重要的是，工程化的“无拇指”细菌 BCCP87 可以被 HCS 生物素化，其底物亲和力恢复到接近正常水平。这项研究表明，细菌羧化酶中的拇指环会干扰与哺乳动物生物素蛋白连接酶的最佳相互作用。虽然拇指环的功能尚不清楚，但这些结果表明了细菌底物对生物素附着的特异性存在限制，而这种限制本身并不是 BirA 的特征。
