The structure of Clostridium perfringens NanI sialidase and its catalytic intermediates
The structure of Clostridium perfringens NanI sialidase and its catalytic intermediates
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DOI：
10.1074/jbc.m710247200
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发表时间：
2008-04-04
影响因子：
4.8
通讯作者：
Taylor, Garry L.
中科院分区：
文献类型：
--
作者：
Newstead, Simon L.;Potter, Jane A.;Taylor, Garry L.
产气荚膜梭菌是一种革兰氏阳性细菌，可引起菌血症、气性坏疽，偶尔也会引起食物中毒。它的基因组编码三种唾液酸酶，nanH， nanI和nanJ，它们参与从各种糖缀合物中去除唾液酸，并在细菌营养和发病机制中发挥作用。最近对锥虫（反式）唾液酸酶的研究表明，所有唾液酸酶的催化作用都可能通过一种类似于其他保留糖苷酶的共价中间体进行。本文通过报道产气荚膜荚膜菌纳米唾液酸酶的催化结构域的0.97埃分辨率的原子结构，及其与底物唾液酸（n-乙酰神经酸）的配合物的0.97 A分辨率，与过渡态类似物（2-脱氧2,3-脱氢- n-乙酰神经酸）的1.5 A分辨率的配合物，以及与使用氟化唾液酸类似物形成的共价中间体的1.2埃分辨率的配合物的配合物，提供了进一步的证据来支持这一建议。总之，这些结构提供了沿催化途径的高分辨率快照。晶体结构表明NanI能够将2-脱氧-2,3-脱氢- n -乙酰神经氨酸水化成n -乙酰神经氨酸。核磁共振证实了这一点，并提出了这种活性的机制。
Clostridium perfringens is a Gram-positive bacterium responsible for bacteremia, gas gangrene, and occasionally food poisoning. Its genome encodes three sialidases, nanH, nanI, and nanJ, that are involved in the removal of sialic acids from a variety of glycoconjugates and that play a role in bacterial nutrition and pathogenesis. Recent studies on trypanosomal ( trans-) sialidases have suggested that catalysis in all sialidases may proceed via a covalent intermediate similar to that of other retaining glycosidases. Here we provide further evidence to support this suggestion by reporting the 0.97 angstrom resolution atomic structure of the catalytic domain of the C. perfringens NanI sialidase, and complexes with its substrate sialic acid ( N-acetylneuramic acid) also to 0.97 A resolution, with a transition-state analogue (2-deoxy2,3-dehydro-N-acetylneuraminic acid) to 1.5 A resolution, and with a covalent intermediate formed using a fluorinated sialic acid analogue to 1.2 angstrom resolution. Together, these structures provide high resolution snapshots along the catalytic pathway. The crystal structures suggested that NanI is able to hydrate 2-deoxy-2,3-dehydro-N-acetylneuraminic acid to N-acetylneuramic acid. This was confirmed by NMR, and a mechanism for this activity is suggested.