Michalska Karolina, Hernandez-Santoyo Alejandra, Jaskolski Mariusz
Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan 60-780, Poland.
J Biol Chem. 2008 May 9;283(19):13388-97. doi: 10.1074/jbc.M800746200. Epub 2008 Mar 10.
Plant l-asparaginases and their bacterial homologs, such as EcAIII found in Escherichia coli, form a subgroup of the N-terminal nucleophile (Ntn)-hydrolase family. In common with all Ntn-hydrolases, they are expressed as inactive precursors that undergo activation in an autocatalytic manner. The maturation process involves intramolecular hydrolysis of a single peptide bond, leading to the formation of two subunits (alpha and beta) folded as one structural domain, with the nucleophilic Thr residue located at the freed N terminus of subunit beta. The mechanism of the autocleavage reaction remains obscure. We have determined the crystal structure of an active site mutant of EcAIII, with the catalytic Thr residue substituted by Ala (T179A). The modification has led to a correctly folded but unprocessed molecule, revealing the geometry and molecular environment of the scissile peptide bond. The autocatalytic reaction is analyzed from the point of view of the Thr(179) side chain rotation, identification of a potential general base residue, and the architecture of the oxyanion hole.
植物L-天冬酰胺酶及其细菌同源物，比如在大肠杆菌中发现的EcAIII，构成了N-末端亲核（Ntn）水解酶家族的一个亚组。与所有Ntn水解酶一样，它们以无活性前体的形式表达，并通过自动催化方式激活。成熟过程涉及单个肽键的分子内水解，导致形成两个折叠成一个结构域的亚基（α和β），亲核苏氨酸残基位于亚基β游离的N末端。自动切割反应的机制仍不清楚。我们已经确定了EcAIII活性位点突变体的晶体结构，其中催化性苏氨酸残基被丙氨酸取代（T179A）。这种修饰导致了一个正确折叠但未加工的分子，揭示了可裂肽键的几何形状和分子环境。从苏氨酸（179）侧链旋转、潜在通用碱基残基的鉴定以及氧负离子洞的结构角度分析了自动催化反应。
