Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo-α-N-acetylgalactosaminidase from Bifidobacterium longum
Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo-α-N-acetylgalactosaminidase from Bifidobacterium longum
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DOI：
10.1074/jbc.m506874200
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发表时间：
2005-11-11
影响因子：
4.8
通讯作者：
Yamamoto, K
中科院分区：
文献类型：
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作者：
Fujita, K;Oura, F;Yamamoto, K
我们在大多数双歧杆菌菌株中发现了内切-α-N-乙酰氨基半乳糖苷酶，这些菌株是人类结肠中的主要细菌。这种酶催化从粘蛋白型糖蛋白中释放α-连接丝氨酸或苏氨酸残基的半乳糖基β 1，3-N-乙酰基-D-半乳糖胺（Gal β 1，3GalNAc）。编码该酶的基因（engBF）已从长双歧杆菌JCM 1217中克隆。该蛋白由1，966个氨基酸残基组成，并且中心结构域（590 - 1381个氨基酸残基）与包括产气荚膜梭菌和肺炎链球菌在内的几种细菌的假设蛋白具有31 - 53%的同一性。在大肠杆菌中表达的重组蛋白从Gal β 1，3GalNAc α 1 pNP和去唾液酸胎球蛋白中释放出Gal β 1，3GalNAc二糖，但不释放GalNAc，Gal β 1，3（GlcNAc β 1，6）GalNAc、GlcNAc β 1，3GalNAc和Gal β 1，3GlcNAc从每种对硝基苯基（pNP）底物中释放，也不从胎球蛋白中释放唾液酸寡糖，表明其对核心1型结构具有严格的底物特异性。水解的立体化学过程由H-1 NMR确定，并发现保留。共22个保守的Asp和Glu残基的定点突变表明，Asp-682和Asp-789是该酶的催化活性的关键残基。该酶还表现出对各种单糖和二糖以及1-烷醇的转糖基化活性，表明其合成新糖缀合物的潜力。这是第一次从任何生物体中分离编码内切-α-N-乙酰氨基半乳糖苷酶的基因，并建立一个新的糖苷水解酶家族（GH家族101）。
We found endo-alpha-N-acetylgalactosaminidase in most bifidobacterial strains, which are predominant bacteria in the human colon. This enzyme catalyzes the liberation of galactosyl beta 1,3-N-acetyl-D-galactosamine (Gal beta 1,3GalNAc) alpha-linked to serine or threonine residues from mucin-type glycoproteins. The gene (engBF) encoding the enzyme has been cloned from Bifidobacterium longum JCM 1217. The protein consisted of 1,966 amino acid residues, and the central domain ( 590 - 1381 amino acid residues) exhibited 31 - 53% identity to hypothetical proteins of several bacteria including Clostridium perfringens and Streptococcus pneumoniae. The recombinant protein expressed in Escherichia coli liberated Gal beta 1,3GalNAc disaccharide from Gal beta 1,3GalNAc alpha 1pNP and asialofetuin, but did not release GalNAc, Gal beta 1,3(GlcNAc beta 1,6) GalNAc, GlcNAc beta 1,3GalNAc, and Gal beta 1,3GlcNAc from each p-nitrophenyl (pNP) substrate, and also did not release sialo-oligosaccharides from fetuin, indicating its strict substrate specificity for the Core 1-type structure. The stereochemical course of hydrolysis was determined by H-1 NMR and was found to be retention. Site-directed mutagenesis of a total of 22 conserved Asp and Glu residues suggested that Asp-682 and Asp-789 are critical residues for the catalytic activity of the enzyme. The enzyme also exhibited transglycosylation activity toward various mono- and disaccharides and 1-alkanols, demonstrating its potential to synthesize neoglycoconjugates. This is the first report for the isolation of a gene encoding endo-alpha-N-acetylgalactosaminidase from any organisms and for the establishment of a new glycoside hydrolase family (GH family 101).