Molecular Basis of 1,6-Anhydro Bond Cleavage and Phosphoryl Transfer by Pseudomonas aeruginosa 1,6-Anhydro-N-acetylmuramic Acid Kinase
Molecular Basis of 1,6-Anhydro Bond Cleavage and Phosphoryl Transfer by Pseudomonas aeruginosa 1,6-Anhydro-N-acetylmuramic Acid Kinase
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DOI：
10.1074/jbc.m110.198317
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发表时间：
2011-04-08
影响因子：
4.8
通讯作者：
Mark, Brian L.
中科院分区：
文献类型：
--
作者：
Bacik, John-Paul;Whitworth, Garrett E.;Mark, Brian L.
脱水-N-乙酰胞壁酸激酶（AnmK）催化革兰氏阴性肽聚糖（PG）再循环中间体1，6-脱水-N-乙酰胞壁酸（anhMurNAc）至N-乙酰胞壁酸-6-磷酸（MurNAc-6-P）的ATP依赖性转化。在这里，我们目前的晶体结构的铜绿假单胞菌AnmK在复杂的天然底物，anhMurNAc，和反应的产物，ADP。AnmK是同源二聚体，每个亚基由两个亚结构域组成，这两个亚结构域由深活性位点裂缝分开，这与属于己糖激酶-hsp 70-肌动蛋白超家族的蛋白质的ATP酶核心相似。anhMurNAc向MurNAc-6-P的转化涉及anhMurNAc的1，6-脱水环的裂解以及沿着向糖的O 6添加磷酰基，因此代表了涉及H3 PO 4正式添加到anhMurNAc的不寻常的酶促机制。反应的结构复合物和NMR分析表明，由Asp-182激活的水分子攻击anhMurNAc的异头碳，有助于1，6-脱水键的裂解，并通过在线磷酰基转移促进O 6对ATP的γ磷酸的捕获。AnmK仅对anhMurNAc有活性，而对代谢相关的1，6-脱水-N-乙酰胞壁酰肽没有活性，这表明在anhMurNAc可以被AnmK作用之前，细胞溶质N-乙酰-脱水胞壁酰-L-丙氨酸酰胺酶AmpD必须首先从这些PG胞壁肽催化剂中除去茎肽。我们的研究为AnmK作为水解酶和一种不寻常的杂环单糖激酶的双重活性的机理模型提供了基础。
Anhydro-N-acetylmuramic acid kinase (AnmK) catalyzes the ATP-dependent conversion of the Gram-negative peptidoglycan (PG) recycling intermediate 1,6-anhydro-N-acetylmuramic acid (anhMurNAc) to N-acetylmuramic acid-6-phosphate (MurNAc-6-P). Here we present crystal structures of Pseudomonas aeruginosa AnmK in complex with its natural substrate, anhMurNAc, and a product of the reaction, ADP. AnmK is homodimeric, with each subunit comprised of two subdomains that are separated by a deep active site cleft, which bears similarity to the ATPase core of proteins belonging to the hexokinase-hsp70-actin superfamily of proteins. The conversion of anhMurNAc to MurNAc-6-P involves both cleavage of the 1,6-anhydro ring of anhMurNAc along with addition of a phosphoryl group to O6 of the sugar, and thus represents an unusual enzymatic mechanism involving the formal addition of H3PO4 to anhMurNAc. The structural complexes and NMR analysis of the reaction suggest that a water molecule, activated by Asp-182, attacks the anomeric carbon of anhMurNAc, aiding cleavage of the 1,6-anhydro bond and facilitating the capture of the gamma phosphate of ATP by O6 via an in-line phosphoryl transfer. AnmK is active only against anhMurNAc and not the metabolically related 1,6-anhydro-N-acetylmuramyl peptides, suggesting that the cytosolic N-acetyl-anhydromuramyl-L-alanine amidase AmpD must first remove the stem peptide from these PG muropeptide catabolites before anhMurNAc can be acted upon by AnmK. Our studies provide the foundation for a mechanistic model for the dual activities of AnmK as a hydrolase and a kinase of an unusual heterocyclic monosaccharide.