Intermolecular cleavage by UmuD-like enzymes: Identification of residues required for cleavage and substrate specificity
Intermolecular cleavage by UmuD-like enzymes: Identification of residues required for cleavage and substrate specificity
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DOI：
10.1006/jmbi.1998.2433
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发表时间：
1999-02-05
影响因子：
5.6
通讯作者：
Woodgate, R
中科院分区：
文献类型：
--
作者：
McDonald, JP;Peat, TS;Woodgate, R
UmuD样蛋白最有特点的是它们在损伤诱导的SOS突变中所起的作用。这一过程中的一个重要步骤是UmuD样蛋白的酶促自我处理。这一反应被认为是通过分子内或分子间自裂解机制发生的。在这里，我们证明它也可以通过异源分子间裂解反应发生。大肠杆菌UmuD酶表现出最广泛的底物专一性，在体内同时裂解大肠杆菌和鼠伤寒沙门氏菌UmuD底物。相比之下，野生型鼠伤寒沙门氏菌UmuD(ST)和粘液A酶催化分子间自切割，但不促进异源切割。然而，对于具有UmuD(ST)残基30-55个残基的嵌合UmuD底物，可以观察到UmuD(ST)酶的异源切割。我们进一步定位了UmuD(ST)催化的异源切割底物分子中Ser50的主要残基。我们假设，该残基的变化通过影响UmuD(ST)酶催化裂解的形成，影响底物分子的裂解位置。这一假说进一步得到以下观察的支持，即已知的破坏大肠杆菌UmuD‘细丝二聚体的突变也阻止了分子间UmuD(EC)的切割。(C)1999年学术出版社。
The UmuD-like proteins are best characterized for their role in damage-induced SOS mutagenesis. An essential step in this process is the enzymatic self-processing of the UmuD-like proteins. This reaction is thought to occur either via an intramolecular or intermolecular self-cleavage mechanism. Here, we demonstrate that it can also occur via an heterologous intermolecular cleavage reaction. The Escherichia coli UmuD enzyme demonstrated the broadest substrate specificity, cleaving both E. coli and Salmonella typhimurium UmuD substrates in vivo. In comparison, the wild-type S. typhimurium UmuD (UmuD(St)) and MucA enzymes catalyzed intermolecular self-cleavage, but did not facilitate heterologous cleavage. Heterologous cleavage by the UmuD(St) enzyme was, however, observed with chimeric UmuD substrates that possess residues 30-55 of UmuD(St). We have further localized the residue predominantly responsible for UmuD(St)-catalyzed heterologous cleavage to Ser50 in the substrate molecule. We hypothesize that changes at this residue affect the positioning of the cleavage site of a substrate molecule within the catalytic cleft of the UmuD(St) enzyme by affecting the formation of a so-called UmuD "filament-dimer". This hypothesis is further supported by the observation that mutations known to disrupt an E. coli UmuD' filament dimer also block intermolecular UmuD(Ec) cleavage. (C) 1999 Academic Press.