Structural and functional determination of homologs of the Mycobacterium tuberculosis N-acetylglucosamine-6-phosphate deacetylase (NagA).
Structural and functional determination of homologs of the Mycobacterium tuberculosis N-acetylglucosamine-6-phosphate deacetylase (NagA).
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DOI：
10.1074/jbc.ra118.002597
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发表时间：
2018-06-22
期刊：
影响因子：
--
通讯作者：
Fullam E
中科院分区：
文献类型：
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作者：
Ahangar MS;Furze CM;Guy CS;Cooper C;Maskew KS;Graham B;Cameron AD;Fullam E
结核分枝杆菌（Mtb）病原体编码属于酰胺水解酶超家族的GlcNAc-6-磷酸脱乙酰酶那牙（Rv 3332）。那牙酶催化GlcNAc-6-磷酸（GlcNAc 6P）脱乙酰化为葡糖胺-6-磷酸（GlcN 6P）。那牙是一个潜在的抗结核药物靶点，因为它代表了Mtb细胞壁生物合成所需的必需氨基糖前体产生的关键酶促步骤，并且还影响细胞壁肽聚糖片段的再循环。在这里，我们报告的结构和功能特性的耻垢分枝杆菌（MSNagA）和海洋分枝杆菌（MMNagA），Mtb的近亲的那牙。使用X-射线晶体学，定点诱变，生物化学和生物物理测定的组合，我们表明，这些分枝杆菌那牙酶的选择性GlcNAc 6P。定点突变研究揭示了活性位点中保守残基的关键作用，这些残基支持底物的立体选择性识别、结合和催化。此外，我们报告的晶体结构的MSNAgA在两个配体的自由形式和复杂的GlcNAc 6P基板在2.6和2.0 μ m的分辨率，分别。GlcNAc 6P复合物结构揭示了GlcNAc 6P的精确结合模式和活性位点的结构框架，包括位于α/β双核位点的两个二价金属。此外，我们观察到位于封闭活性位点的柔性环区域上的半胱氨酸残基。这种半胱氨酸是分枝杆菌所独有的，可能代表了靶向分枝杆菌那牙酶的独特亚位点。我们的研究结果提供了重要的见解的结构和机械特性的分枝杆菌那牙酶具有重要作用的氨基糖和核苷酸代谢的分枝杆菌。
The Mycobacterium tuberculosis (Mtb) pathogen encodes a GlcNAc-6-phosphate deacetylase enzyme, NagA (Rv3332), that belongs to the amidohydrolase superfamily. NagA enzymes catalyze the deacetylation of GlcNAc-6-phosphate (GlcNAc6P) to glucosamine-6-phosphate (GlcN6P). NagA is a potential antitubercular drug target because it represents the key enzymatic step in the generation of essential amino-sugar precursors required for Mtb cell wall biosynthesis and also influences recycling of cell wall peptidoglycan fragments. Here, we report the structural and functional characterization of NagA from Mycobacterium smegmatis (MSNagA) and Mycobacterium marinum (MMNagA), close relatives of Mtb. Using a combination of X-ray crystallography, site-directed mutagenesis, and biochemical and biophysical assays, we show that these mycobacterial NagA enzymes are selective for GlcNAc6P. Site-directed mutagenesis studies revealed crucial roles of conserved residues in the active site that underpin stereoselective recognition, binding, and catalysis of substrates. Moreover, we report the crystal structure of MSNagA in both ligand-free form and in complex with the GlcNAc6P substrate at 2.6 and 2.0 Å resolutions, respectively. The GlcNAc6P complex structure disclosed the precise mode of GlcNAc6P binding and the structural framework of the active site, including two divalent metals located in the α/β binuclear site. Furthermore, we observed a cysteine residue located on a flexible loop region that occludes the active site. This cysteine is unique to mycobacteria and may represent a unique subsite for targeting mycobacterial NagA enzymes. Our results provide critical insights into the structural and mechanistic properties of mycobacterial NagA enzymes having an essential role in amino-sugar and nucleotide metabolism in mycobacteria.