Identification and characterization of important residues in the catalytic mechanism of CMP-Neu5Ac synthetase from Neisseria meningitidis.
Identification and characterization of important residues in the catalytic mechanism of CMP-Neu5Ac synthetase from Neisseria meningitidis.
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DOI：
10.1111/j.1742-4658.2010.07696.x
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发表时间：
2010-07
期刊：
影响因子：
--
通讯作者：
Berry A
中科院分区：
文献类型：
--
作者：
Horsfall LE;Nelson A;Berry A
唾液酸化低聚糖存在于哺乳动物细胞外表面，在细胞相互作用中起着至关重要的作用，一些细菌能够模仿这些结构来逃避宿主的免疫系统。这将是非常有益的研究感染性和自身免疫性疾病和癌症，详细了解唾液酸化的途径，使抑制剂和模拟物的设计和生产。唾液酸化分两个阶段进行，第一阶段激活唾液酸，第二阶段将其转移到靶分子。活化步骤由酶CMP-Neu 5Ac合成酶（CNS）催化。在这里，我们使用CNS和类似酶的晶体结构来预测脑膜炎奈瑟菌CNS中重要的残基。9个残基突变为丙氨酸，并使用连续测定法测量稳态酶动力学参数，以检测反应产物之一焦磷酸。导致活性损失最大的突变包括K142 A、D211 A、D209 A和残基Q104处的一系列突变，从相关酶的序列比对研究中突出显示，表明这些残基在CNS的催化机制中具有重要作用。D211 A和D209 A的突变为先前提出的酶中的金属结合位点提供了强有力的证据，并且我们在残基Q104处的突变的结果使我们将该残基包括在中间复合物的金属结合位点中。这表明，像糖活化脂多糖合成CMP-2-酮-3-脱氧-甘露-辛酸合成酶KdsB一样，CNS在催化循环期间募集两个Mg 2+离子。
Sialylated oligosaccharides, present on mammalian outer-cell surfaces, play vital roles in cellular interactions and some bacteria are able to mimic these structures to evade their host’s immune system. It would be of great benefit to the study of infectious and autoimmune diseases and cancers, to understand the pathway of sialylation in detail to enable the design and production of inhibitors and mimetics. Sialylation occurs in two stages, the first to activate sialic acid and the second to transfer it to the target molecule. The activation step is catalysed by the enzyme CMP-Neu5Ac synthetase (CNS). Here we used crystal structures of CNS and similar enzymes to predict residues of importance in the CNS from Neisseria meningitidis. Nine residues were mutated to alanine, and the steady-state enzyme kinetic parameters were measured using a continuous assay to detect one of the products of the reaction, pyrophosphate. Mutations that caused the greatest loss in activity included K142A, D211A, D209A and a series of mutations at residue Q104, highlighted from sequence-alignment studies of related enzymes, demonstrating significant roles for these residues in the catalytic mechanism of CNS. The mutations of D211A and D209A provide strong evidence for a previously proposed metal-binding site in the enzyme, and the results of our mutations at residue Q104 lead us to include this residue in the metal-binding site of an intermediate complex. This suggests that, like the sugar-activating lipopolysaccharide-synthesizing CMP-2-keto-3-deoxy-manno-octonic acid synthetase enzyme KdsB, CNS recruits two Mg2+ ions during the catalytic cycle.
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影响因子：
56.9
作者：
FLEISCHMANN, RD;ADAMS, MD;VENTER, JC
通讯作者：
VENTER, JC
影响因子：
4.3
作者：
Nakata, D;Münster, AK;Kitajima, K
通讯作者：
Kitajima, K
影响因子：
4.8
作者：
Tullius, MV;Munson, RS;Gibson, BW
通讯作者：
Gibson, BW
影响因子：
4.8
作者：
Münster, AK;Weinhold, B;Gerardy-Schahn, R
通讯作者：
Gerardy-Schahn, R
影响因子：
3.6
作者：
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通讯作者：
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