A structural factor responsible for substrate recognition by Bacillus sp. GL1 xanthan lyase that acts specifically on pyruvated side chains of xanthan.
A structural factor responsible for substrate recognition by Bacillus sp. GL1 xanthan lyase that acts specifically on pyruvated side chains of xanthan.
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DOI：
10.1021/bi0619775
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发表时间：
2007-01
期刊：
影响因子：
2.9
通讯作者：
Y. Maruyama;B. Mikami;W. Hashimoto;K. Murata
中科院分区：
文献类型：
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作者：
Y. Maruyama;B. Mikami;W. Hashimoto;K. Murata
黄原胶是由五糖重复单元组成的细菌杂多糖，即，纤维二糖作为主链，由两个甘露糖和一个葡糖醛酸组成的三糖作为侧链。黄原胶侧链的非还原性末端甘露糖残基被部分取代。芽孢杆菌GL 1黄原胶裂解酶是多糖裂解酶家族8的成员，其特异性地作用于黄原胶的变性侧链，并通过使用单个Tyr 255残基作为碱和酸催化剂的β-消除反应产生变性甘露糖。在这里，我们通过X-射线晶体学和突变分析显示黄原胶裂解酶底物识别的结构因素。该酶在-1亚位点上容纳甘露糖和双甘露糖，尽管这两种单糖的抑制剂和解离常数表明双甘露糖对黄原胶裂解酶的亲和力远高于甘露糖。还原甘露糖的高亲和力可能是由于还原甘露糖的羧基与Tyr 315和Arg 612的氨基酸残基之间形成了额外的氢键。对这两个残基的定点突变表明，Arg 612是识别双甘露糖的关键残基。Arg 612位于覆盖底物的突出环中，表明该环起着盖子的作用，负责在活性位点处适当地容纳底物。
Xanthan is a bacterial heteropolysaccharide composed of pentasaccharide repeating units, i.e., a cellobiose as a backbone and a trisaccharide consisting of two mannoses and one glucuronic acid as a side chain. Nonreducing terminal mannose residues of xanthan side chains are partially pyruvated. Bacillus sp. GL1 xanthan lyase, a member of polysaccharide lyase family 8, acts specifically on pyruvated side chains of xanthan and yields pyruvated mannose through a beta-elimination reaction by using a single Tyr255 residue as base and acid catalysts. Here we show structural factors for substrate recognition by xanthan lyase through X-ray crystallographic and mutational analyses. The enzyme accommodates mannose and pyruvated mannose at the -1 subsite, although both inhibitor and dissociation constants of the two monosaccharides indicated that the affinity of pyruvated mannose for xanthan lyase is much higher than that of mannose. The high affinity of pyruvated mannose is probably due to the formation of additional hydrogen bonds between the carboxyl group of pyruvated mannose and amino acid residues of Tyr315 and Arg612. Site-directed mutagenesis of the two residues demonstrated that Arg612 is a key residue in recognizing pyruvated mannose. Arg612 is located in the protruding loop covering the substrate, suggesting that the loop functions as a lid that is responsible for the proper accommodation of the substrate at the active site.