Evolution and Biochemistry of Family 4 Glycosidases: Implications for Assigning Enzyme Function in Sequence Annotations
Evolution and Biochemistry of Family 4 Glycosidases: Implications for Assigning Enzyme Function in Sequence Annotations
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DOI：
10.1093/molbev/msp162
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发表时间：
2009-11-01
影响因子：
10.7
通讯作者：
Thompson, John
中科院分区：
文献类型：
--
作者：
Hall, Barry G.;Pikis, Andreas;Thompson, John
糖基水解酶家族4（GH4）是该酶超家族中114个家族中的一个例外。GH4的成员表现出不寻常的活性辅因子的要求，和一个必需的半胱氨酸残基是目前在活性位点。最重要的是，GH4的成员采用独特的催化机制来裂解糖苷键。通过系统发育分析，并从可用的底物特异性，我们已经分配了大多数的酶的GH4到五个亚组。我们的分类揭示了底物特异性与每个亚组中存在的四个氨基酸基序之间的意想不到的关系，所述基序包括活性位点Cys残基：α-葡糖苷酶，CHE（I/V）; α-半乳糖苷酶，CHSV; α-葡糖醛酸糖苷酶，CHGx; 6-磷酸-α-葡糖苷酶，CsA;和6-磷酸-β-葡糖苷酶，CN（V/I）P。一个特定的基序的存在是否足以预测一个未分配的GH4蛋白的催化功能？为了验证这一假设，我们已经从植物病原体大黄欧文氏菌纯化并表征了α-葡糖苷特异性GH4酶（PalH）。通过定点突变改变了该蛋白质中的CHEI基序，并确定了对底物特异性的影响。CHSV的变化导致所有α-葡糖苷酶活性的丧失，但突变蛋白质未表现出预期的α-半乳糖苷酶活性。含半胱氨酸的基序可能暗示酶的特异性，但系统发育的位置需要在该特异性的信心。莱氏无胆甾原体GH4蛋白在遗传学上是磷酸-β-葡糖苷酶，但具有独特的SSSP基序。在该基序中缺乏初始Cys，它不能通过正常的GH4机制水解糖苷，因为需要Cys定位金属离子以进行水解，它也不能使用更常见的Koshland单或双置换机制。几个考虑表明，蛋白质已经获得了一个新的功能，积极选择的结果。这项研究强调了自动注释系统的重要性，通过整合系统发育分析，功能基序和生物信息学数据，可能会导致创新的实验，进一步我们对生物系统的理解。
Glycosyl hydrolase Family 4 (GH4) is exceptional among the 114 families in this enzyme superfamily. Members of GH4 exhibit unusual cofactor requirements for activity, and an essential cysteine residue is present at the active site. Of greatest significance is the fact that members of GH4 employ a unique catalytic mechanism for cleavage of the glycosidic bond. By phylogenetic analysis, and from available substrate specificities, we have assigned a majority of the enzymes of GH4 to five subgroups. Our classification revealed an unexpected relationship between substrate specificity and the presence, in each subgroup, of a motif of four amino acids that includes the active-site Cys residue: a-glucosidase, CHE(I/V); alpha-galactosidase, CHSV; alpha-glucuronidase, CHGx; 6-phospho-alpha-glucosidase, CDMP; and 6-phospho-beta-glucosidase, CN(V/I)P. The question arises: Does the presence of a particular Motif sufficiently predict the catalytic function of an unassigned GH4 protein? To test this hypothesis, we have purified and characterized the a-glucoside -specific GH4 enzyme (PalH) from the phytopathogen, Erwinia rhapontici. The CHEI motif in this protein has been changed by site-dirceted mutagenesis, and the effects upon Substrate specificity have been determined. The change to CHSV caused the loss of all a-glucosidase activity, but the mutant protein exhibited none of the anticipated alpha-galactosidase activity. The Cys-containing motif may be suggestive of enzyme specificity, but phylogenetic placement is required for confidence in that specificity. The Acholeplasma laidlawii GH4 protein is phylogenetically a phospho-beta-glucosidase but has a unique SSSP motif. Lacking the initial Cys in that motif it cannot hydrolyze glycosides by the normal GH4 mechanism because the Cys is required to position the metal ion for hydrolysis, nor can it use the more common single or double-displacement mechanism of Koshland. Several considerations suggest that the protein has acquired a new function as the consequence of positive selection. This study emphasizes the importance of automatic annotation systems that by integrating phylogenetic analysis, functional motifs, and bioinformatics data, may lead to innovative experiments that further our understanding of biological systems.