Amino acid 310 determines the donor substrate specificity of serogroup W-135 and Y capsule polymerases of Neisseria meningitidis
Amino acid 310 determines the donor substrate specificity of serogroup W-135 and Y capsule polymerases of Neisseria meningitidis
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DOI：
10.1111/j.1365-2958.2008.06580.x
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发表时间：
2009-02-01
影响因子：
3.6
通讯作者：
Vogel, Ulrich
中科院分区：
文献类型：
--
作者：
Claus, Heike;Stummeyer, Katharina;Vogel, Ulrich
血清群W-135和Y脑膜炎球菌的荚膜多糖是含唾液酸的杂聚物，第二个糖残基是半乳糖或葡萄糖。如前所示，预测的催化胶囊聚合的酶，即SiaD（W-135）和SiaD（Y）的序列仅在几个氨基酸上不同。通过用纯化的重组蛋白进行体外测定，现已证实SiaD（W-135）和SiaD（Y）是具有己糖基转移酶和唾液酸转移酶活性的胶囊聚合酶。为了鉴定对胶囊聚合酶的底物特异性至关重要的氨基酸，通过DNA序列比较和随后的定点诱变来缩小多态性位点。血清群特异性氨基酸仅限于蛋白质的N-末端部分。如免疫化学和体外活性测定所示，位于酶预测的己糖基转移酶部分的核苷酸识别结构域内的氨基酸310是唯一的底物特异性。Pro-310确定产生血清群W-135荚膜的半乳糖基转移酶活性，Gly-310确定产生血清群Y荚膜的葡糖基转移酶活性。计算机模拟分析显示，在相同糖基转移酶家族的其他成员中，无论细菌种属如何，都存在类似的基于氨基酸的关联。
The capsular polysaccharides of serogroup W-135 and Y meningococci are sialic acid-containing heteropolymers, with either galactose or glucose as the second sugar residue. As shown previously, sequences of the predicted enzymes that catalyse capsule polymerization, i.e. SiaD(W-135) and SiaD(Y), differ in only a few amino acids. By in vitro assays with purified recombinant proteins, SiaD(W-135) and SiaD(Y) were now confirmed to be the capsule polymerases harbouring both hexosyltransferase and sialyltransferase activity. In order to identify amino acids crucial for substrate specificity of the capsule polymerases, polymorphic sites were narrowed down by DNA sequence comparisons and subsequent site-directed mutagenesis. Serogroup-specific amino acids were restricted to the N-terminal part of the proteins. Exclusively amino acid 310, located within the nucleotide recognition domain of the enzymes' predicted hexosyltransferase moiety, accounted for substrate specificity as shown by immunochemistry and in vitro activity assay. Pro-310 determined galactosyltransferase activity that resulted in a serogroup W-135 capsule and Gly-310 determined glucosyltransferase activity that resulted in a serogroup Y capsule. In silico analysis revealed a similar amino acid-based association in other members of the same glycosyltransferase family irrespective of the bacterial species.