Deciphering the molecular basis of the broad substrate specificity of alpha-glucosidase from Bacillus sp. SAM1606.
Deciphering the molecular basis of the broad substrate specificity of alpha-glucosidase from Bacillus sp. SAM1606.
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破译芽孢杆菌属α-葡萄糖苷酶广泛底物特异性的分子基础。
DOI：
10.1093/jb/mvg172
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发表时间：
2003
影响因子：
2.7
通讯作者：
T. Nishino
中科院分区：
文献类型：
--
作者：
Akio Noguchi;Masayasu Yano;Y. Ohshima;H. Hemmi;M. Inohara;M. Okada;K. Min;T. Nakayama;T. Nishino
芽孢杆菌属菌株SAM 1606的α-葡萄糖苷酶是糖基水解酶家族13的成员，具有非常广泛的底物特异性，是极少数能够有效水解α，α '-海藻糖（海藻糖）的α-1，1-糖苷键的α-葡萄糖苷酶之一。家族-13酶的系统发育分析表明，SAM 1606 α-葡萄糖苷酶可能是从α-1，6-特异性祖先，寡-1，6-葡萄糖苷酶（O 16 G）进化而来。事实上，替换B的Pro（273*）和Thr（342*）。cereus O 16 G通过甘氨酸和天冬酰胺（SAM 1606酶中的相应残基）分别导致海藻糖的相对催化效率提高192倍，这表明O 16 G可以通过取代有限数量的氨基酸而容易地“进化”成具有扩展的底物特异性的酶，包括在位置273*（星号表示SAM 1606序列的氨基酸编号）。为了探测α-葡糖苷酶273* 位氨基酸在确定底物特异性中的作用，将SAM 1606 α-葡糖苷酶273位氨基酸替换为所有其它天然存在的氨基酸，并对所得突变体进行动力学表征。结果表明，大体积残基的取代（例如，异亮氨酸和甲硫氨酸）取代甘氨酸导致海藻糖和麦芽糖的K（m）值大幅增加，而对异麦芽糖的亲和力仅受该位置的这种氨基酸取代的最小影响。构建了野生型和突变型SAM 1606 α-葡糖苷酶的酶-底物复合物的三维结构模型，以探索导致这些观察结果的机制。有人提出，在位置273* 甘氨酸取代可以消除周围的空间位阻+1，原来发生在亲本O 16 G，是，至少部分，负责获得广泛的底物特异性SAM 1606 α-葡萄糖苷酶。
The alpha-glucosidase of Bacillus sp. strain SAM1606 is a member of glycosyl hydrolase family 13, and shows an extraordinarily broad substrate specificity and is one of very few alpha-glucosidases that can efficiently hydrolyze the alpha-1,1-glucosidic linkage of alpha,alpha'-trehalose (trehalose). Phylogenetic analysis of family-13 enzymes suggests that SAM1606 alpha-glucosidase may be evolutionally derived from an alpha-1,6-specific ancestor, oligo-1,6-glucosidase (O16G). Indeed, replacement of Pro(273*) and Thr(342*) of B. cereus O16G by glycine and asparagine (the corresponding residues in the SAM1606 enzyme), respectively, was found to cause 192-fold enhancement of the relative catalytic efficiency for trehalose, suggesting that O16G may easily "evolved" into an enzyme with an extended substrate specificity by substitution of a limited number of amino acids, including that at position 273* (an asterisk indicates the amino-acid numbering of the SAM1606 sequence). To probe the role of the amino acid at position 273* of alpha-glucosidase in determination of the substrate specificity, the amino acid at position 273 of SAM1606 alpha-glucosidase was replaced by all other naturally occurring amino acids, and the resultant mutants were kinetically characterized. The results showed that substitution of bulky residues (e.g., isoleucine and methionine) for glycine at this position resulted in large increases in the K(m) values for trehalose and maltose, whereas the affinity to isomaltose was only minimally affected by such an amino-acid substitution at this position. Three-dimensional structural models of the enzyme-substrate complexes of the wild-type and mutant SAM1606 alpha-glucosidases were built to explore the mechanism responsible for these observations. It is proposed that substitution by glycine at position 273* could eliminate steric hindrance around subsite +1 that originally occurred in parental O16G and is, at least in part, responsible for the acquired broad substrate specificity of SAM1606 alpha-glucosidase.