Mutational analysis of peptidoglycan amidase MepA
Mutational analysis of peptidoglycan amidase MepA
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DOI：
10.1021/bi0613776
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发表时间：
2007-01-09
期刊：
影响因子：
2.9
通讯作者：
Bochtler, Matthias
中科院分区：
文献类型：
--
作者：
Firczuk, Malgorzata;Bochtler, Matthias
大肠杆菌蛋白内肽酶A （MepA）是一种降解大肠杆菌肽聚糖中D-丙氨酸和中-2,6-二氨基戊酸之间D，D酰胺键的酶。MepA及其在其他变形菌中的同源物与D-Ala-D-Ala金属肽酶在整体结构上具有相似性，在活性位点附近与溶葡萄蛋白型酶具有局部相似性，这促使这些酶被分类为LAS酶。LAS酶的活性位点含有一个二价阳离子，其晶体结构为四配位。三种金属配体在所有结构上都是相同的，但第四种配体的身份不同。靠近金属的两个残基可能充当一般的酸/碱，但它们的作用尚不清楚。在这里，我们报道了一种新的MepA表达系统，它允许从内源性野生型酶中分离MepA变体，并使用定义的肽聚糖片段进行HPLC分析，该分析允许评估MepA活性而无需重新折叠步骤。我们发现保守的金属配体是折叠（D120）或催化（H113, H211）所必需的。候选催化残基H206或H209和“第四”金属配体H110的单独突变对折叠是耐受的，但会大大降低活性。残基W203突变为天冬氨酸损害底物结合。
Murein endopeptidase A (MepA) from Escherichia coli is a periplasmic peptidoglycan amidase that cleaves D,D amide bonds between D-alanine and meso-2,6-diaminopimelic acid in E. coli peptidoglycan. MepA and its homologues in other proteobacteria share overall structural similarity with D-Ala-D-Ala metallopeptidases and local similarity around the active site with lysostaphin-type enzymes, which has prompted the classification of these enzymes as LAS enzymes. LAS enzymes contain a single divalent cation in the active site, which is tetracoordinated in the crystal structures. Three of the metal ligands are identical in all structures, but the identity of the fourth ligand varies. Two residues in proximity to the metal might act as a general acid/base, but their role is not clear. Here, we report a new MepA expression system, which allows the separation of MepA variants from the endogenous wild-type enzyme, and an HPLC assay with a defined peptidoglycan fragment, which allows assessment of MepA activity without a refolding step. We find that the conserved metal ligands are required for folding (D120) or catalysis (H113, H211). Separate mutations of the candidate catalytic residues H206 or H209 and of the "fourth" metal ligand H110 are tolerated for folding but drastically reduce activity. Mutation of residue W203 to aspartate impairs substrate binding.