The structure of hyperthermophilic β-N-acetylglucosaminidase reveals a novel dimer architecture associated with the active site.
The structure of hyperthermophilic β-N-acetylglucosaminidase reveals a novel dimer architecture associated with the active site.
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超嗜热 β-N-乙酰氨基葡萄糖苷酶的结构揭示了与活性位点相关的新型二聚体结构。
DOI：
10.1111/febs.13049
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发表时间：
2014
期刊：
影响因子：
5.4
通讯作者：
Tsuyoshi Inoue and Kazuhiko Ishikawa
中科院分区：
文献类型：
--
作者：
Shouhei Mine;Yuji Kado;Masahiro Watanabe;Yohta Fukuda;Yoshito Abe;Tadashi Ueda;Yutaka Kawarabayasi;Tsuyoshi Inoue and Kazuhiko Ishikawa
嗜热菌β-N-乙酰氨基葡萄糖苷酶(NAGA)能将壳低聚物降解成单体β-N-乙酰氨基葡萄糖。虽然NAGA在糖苷水解酶(GH)家族3中含有一个高度保守的序列基序，但它的底物专一性和生物组装特性使其有别于其他GH家族3β-N-乙酰氨基葡萄糖苷酶。为了研究其活性中心附近的独特结构，我们测定了NaGA的晶体结构，分辨率为2.43。NAGA形成二聚体结构，其中单体结构由N-末端结构域和C-末端结构域组成。该二聚体结构对形成二聚体具有较高的溶剂化自由能。通过诱变分析，推测催化亲核体和一般酸碱残基分别为Asp245和His173。NAGA最显著的特点是在下一个多肽链的N-末端结构域和C-末端结构域之间形成活性部位裂隙，而其他两个结构域的GH家族3酶则形成同一分子内的活性部位。另一个显著的特点是，位于活动场地周围的环路显示出很高的灵活性。其中一个柔性环含有一般的酸碱His173，被认为与催化过程中的底物扭曲有关。此外，与活性中心密切接触的环来自下一个多肽链的C-末端结构域，包含一个高B因子值的区域，表明C-末端结构域参与催化的可能性。这些结果表明，NAGA的二聚体结构对其活性和热稳定性是重要的。数据库结构数据可在蛋白质数据库中获得，登录号为3WO8。结构化数字摘要NAGA和NAGA可通过X射线结晶学结合(查看交互作用)
The β‐N‐acetylglucosaminidase from the hyperthermophilic bacteriaThermotoga maritima(NagA) hydrolyzes chitooligomers into monomer β‐N‐acetylglucosamine. Although NagA contains a highly conserved sequence motif found in glycoside hydrolase (GH) family 3, it can be distinguished from other GH family 3 β‐N‐acetylglucosaminidases by its substrate specificity and biological assembly. To investigate its unique structure around the active site, we determined the crystal structure of NagA at a resolution of 2.43 Å. The NagA forms a dimer structure in which the monomer structure consists of an N‐ and a C‐terminal domain. The dimer structure exhibits high solvation free energy for dimer formation. From mutagenesis analyses, the catalytic nucleophile and general acid–base residues were supposed to be Asp245 and His173, respectively. The most striking characteristic of NagA was that it forms the active site cleft from the N‐terminal domain and the C‐terminal domain of the next polypeptide chain, whereas the other two‐domain GH family 3 enzymes form the site within the same molecule. Another striking feature is that the loops located around the active site show high flexibility. One of the flexible loops contains the general acid–base His173 and was thought to be involved in substrate distortion during catalysis. In addition, a loop in close contact with the active site, which comes from the C‐terminal domain of the next polypeptide chain, contains a region of highB‐factor values, indicating the possibility that the C‐terminal domain is involved in catalysis. These results suggest that the dimer structure of NagA is important for its activity and thermostability.DatabaseStructural data are available in the Protein Data Bank under accession number 3WO8.Structured digital abstractNagA and NagA bind by x-ray crystallography (View interaction)