Meekrathok Piyanat, Suginta Wipa
Biochemistry-Electrochemistry Research Unit and School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand.
Center of Excellence in Advanced Functional Materials, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand.
PLoS One. 2016 Feb 12;11(2):e0149228. doi: 10.1371/journal.pone.0149228. eCollection 2016.
Vibrio harveyi GH20 β-N-acetylglucosaminidase (VhGlcNAcase) is a chitinolytic enzyme responsible for the successive degradation of chitin fragments to GlcNAc monomers, activating the onset of the chitin catabolic cascade in marine Vibrios.
Two invariant acidic pairs (Asp303-Asp304 and Asp437-Glu438) of VhGlcNAcase were mutated using a site-directed mutagenesis strategy. The effects of these mutations were examined and the catalytic roles of these active-site residues were elucidated using a chemical rescue approach. Enhancement of the enzymic activity of the VhGlcNAcase mutants was evaluated by a colorimetric assay using pNP-GlcNAc as substrate.
Substitution of Asp303, Asp304, Asp437 or Glu438 with Ala/Asn/Gln produced a dramatic loss of the GlcNAcase activity. However, the activity of the inactive D437A mutant was recovered in the presence of sodium formate. Our kinetic data suggest that formate ion plays a nucleophilic role by mimicking the β-COO-side chain of Asp437, thereby stabilizing the reaction intermediate during both the glycosylation and the deglycosylation steps.
Chemical rescue of the inactive D437A mutant of VhGlcNAcase by an added nucleophile helped to identify Asp437 as the catalytic nucleophile/base, and hence its acidic partner Glu438 as the catalytic proton donor/acceptor.
Identification of the catalytic nucleophile of VhGlcNAcases supports the proposal of a substrate-assisted mechanism of GH20 GlcNAcases, requiring the catalytic pair Asp437-Glu438 for catalysis. The results suggest the mechanistic basis of the participation of β-N-acetylglucosaminidase in the chitin catabolic pathway of marine Vibrios.
哈维氏弧菌GH20β-N-乙酰氨基葡萄糖苷酶（VhGlcNAcase）是一种几丁质分解酶，负责将几丁质片段连续降解为N-乙酰葡糖胺单体，从而启动海洋弧菌中几丁质分解代谢级联反应。
采用定点诱变策略对VhGlcNAcase的两对不变酸性氨基酸对（Asp303-Asp304和Asp437-Glu438）进行突变。检测这些突变的影响，并使用化学拯救方法阐明这些活性位点残基的催化作用。以对硝基苯-N-乙酰-β-D-氨基葡萄糖（pNP-GlcNAc）为底物，通过比色法评估VhGlcNAcase突变体酶活性的增强情况。
用丙氨酸/天冬酰胺/谷氨酰胺取代Asp303、Asp304、Asp437或Glu438会导致N-乙酰氨基葡萄糖苷酶活性急剧丧失。然而，在甲酸钠存在的情况下，无活性的D437A突变体的活性得以恢复。我们的动力学数据表明，甲酸根离子通过模拟Asp437的β-COO侧链发挥亲核作用，从而在糖基化和去糖基化步骤中稳定反应中间体。
通过添加亲核试剂对无活性的VhGlcNAcase D437A突变体进行化学拯救，有助于确定Asp437为催化亲核试剂/碱，因此其酸性伙伴Glu438为催化质子供体/受体。
确定VhGlcNAcases的催化亲核试剂支持了GH20 N-乙酰氨基葡萄糖苷酶底物辅助机制的提议，该机制催化需要Asp437-Glu438这一催化对。结果表明了β-N-乙酰氨基葡萄糖苷酶参与海洋弧菌几丁质分解代谢途径的机制基础。
