Structural and functional characterization of a glycoside hydrolase family 3 β-N-acetylglucosaminidase from Paenibacillus sp. str. FPU-7
Structural and functional characterization of a glycoside hydrolase family 3 β-N-acetylglucosaminidase from Paenibacillus sp. str. FPU-7
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DOI：
10.1093/jb/mvz072
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发表时间：
2019-12-01
影响因子：
2.7
通讯作者：
Kimoto, Hisashi
中科院分区：
文献类型：
--
作者：
Itoh, Takafumi;Araki, Tomomitsu;Kimoto, Hisashi
甲壳素是一种β-1，4-连接的N-乙酰-D-氨基葡萄糖(GlcNAc)均多糖，是地球上含量最丰富的生物聚合物之一。拟芽孢杆菌属Str.FPU-7产生几种不同的几丁质酶，并在培养基中将几丁质转化为N，N‘-二乙酰基壳二糖((GlcNAc)2)。然而，芽孢杆菌将(GlcNAc)2输入细胞质并将其分解为单体GlcNAc的机制尚不清楚。在拟芽孢杆菌中发现了编码N-乙酰-D-氨基葡萄糖苷酶(PsNagA)的基因。Str.使用表达克隆系统获得了FPU-7基因组。推定的PsNagA氨基酸序列表明该酶属于糖苷水解酶家族3(GH3)。成功地在大肠杆菌中高效表达了重组PsNagA，并进行了纯化。经凝胶渗透色谱鉴定，该酶以57 kDa的单体形式存在。PsNagA特异性地降解甲壳素低聚糖(GlcNAc)(2-4)、4-硝基苯基N-乙酰β-D-氨基葡萄糖(PNP-GlcNAc)和PNP-(GlcNAc)(2-6)，但对4-硝基苯基-β-D-葡萄糖、4-硝基苯基-β-D-半乳糖胺和胶体甲壳素没有检测到的活性。在本研究中，我们提出了与GlcNAc结合的PsNagA的1.9埃晶体结构。晶体结构揭示了与底物识别相关的结构特征和PsNagA的催化机理。本研究首次对拟青霉GH3β-N-乙酰-D-氨基葡萄糖苷酶的结构和功能进行了研究。
Chitin, a beta-1,4-linked homopolysaccharide of N-acetyl-D-glucosamine (GlcNAc), is one of the most abundant biopolymers on Earth. Paenibacillus sp. str. FPU-7 produces several different chitinases and converts chitin into N,N'-diacetylchitobiose ((GlcNAc)2) in the culture medium. However, the mechanism by which the Paenibacillus species imports (GlcNAc) 2 into the cytoplasm and divides it into the monomer GlcNAc remains unclear. The gene encoding Paenibacillus beta-N-acetyl-D-glucosaminidase (PsNagA) was identified in the Paenibacillus sp. str. FPU-7 genome using an expression cloning system. The deduced amino acid sequence of PsNagA suggests that the enzyme is a part of the glycoside hydrolase family 3 (GH3). Recombinant PsNagA was successfully overexpressed in Escherichia coli and purified to homogeneity. As assessed by gel permeation chromatography, the enzyme exists as a 57-kDa monomer. PsNagA specifically hydrolyses chitin oligosaccharides, (GlcNAc)(2-4), 4-nitrophenyl N-acetyl beta-D-glucosamine (pNP-GlcNAc) and pNP-(GlcNAc)(2-6), but has no detectable activity against 4-nitrophenyl beta-D-glucose, 4-nitrophenyl beta-D-galactosamine and colloidal chitin. In this study, we present a 1.9 angstrom crystal structure of PsNagA bound to GlcNAc. The crystal structure reveals structural features related to substrate recognition and the catalytic mechanism of PsNagA. This is the first study on the structural and functional characterization of a GH3 beta-N-acetyl-D-glucosaminidase from Paenibacillus sp.