Altering substrate specificity of Bacillus sp SAM1606 alpha-glucosidase by comparative site-specific mutagenesis
Altering substrate specificity of Bacillus sp SAM1606 alpha-glucosidase by comparative site-specific mutagenesis
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DOI：
10.1074/jbc.272.3.1601
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发表时间：
1997-01-17
影响因子：
4.8
通讯作者：
Shibano, Y
中科院分区：
文献类型：
--
作者：
InoharaOchiai, M;Nakayama, T;Shibano, Y
芽孢杆菌属 sp. SAM1606 α-葡萄糖苷酶具有广泛的底物特异性，是唯一已知的能够有效水解 α,α'-海藻糖的 α-葡萄糖苷酶。该酶与热葡萄糖芽孢杆菌和蜡状芽孢杆菌的寡聚 1,6-葡萄糖苷酶 (O16G) 具有非常高的序列相似性，不能作用于海藻糖。这三种酶在保守区 (CR) 内具有 80% 相同的残基，这些残基被认为位于 α-淀粉酶家族酶的活性位点附近或处。为了通过定点诱变鉴定决定 SAM1606 酶广泛底物特异性的关键残基，我们比较了这三种葡萄糖苷酶的 CR 序列，并选择了 SAM1606 α-葡萄糖苷酶中要诱变的 5 个靶标：Met (76)、Arg (81)、Ala (116)、Gly (273) 和 Thr (342) 这些残基已被专门替换 分别用 Asn、Ser、Val、Pro 和 Asn 进行体外诱变，如芽孢杆菌 O16G 中那样。对具有单取代和多取代的 12 种突变酶进行了表达和动力学表征。结果表明，5 倍突变实际上消除了该酶对 α,α'-海藻糖的亲和力，而异麦芽糖（SAM1606 酶和 O16G 的良好底物）水解的特异性常数在突变后基本保持不变。海藻糖亲和力的丧失主要受到 Gly(273) --> Pro 取代的控制，其效果通过 B 倍和四重突变体中的 Thr(342) --> Asn 取代而特别增强。这些结果为 CR 中的氨基酸残基在确定 α-葡萄糖苷酶的底物特异性中的不同作用提供了证据。
The Bacillus sp. SAM1606 alpha-glucosidase with a broad substrate specificity is the only known alpha-glucosidase that can hydrolyze alpha,alpha'-trehalose efficiently. The enzyme exhibits a very high sequence similarity to the oligo 1,6-glucosidases (O16G) of Bacillus thermoglucosidasius and Bacillus cereus which cannot act on trehalose. These three enzymes share 80% identical residues within the conserved regions (CR), which have been suggested to be located near or at the active site of the alpha-amylase family enzymes. To identify by site-specific mutagenesis the critical residues that determine the broad substrate specificity of the SAM1606 enzyme we compared the CR sequences of these three glucosidases and selected five targets to be mutagenized in SAM1606 alpha-glucosidase, Met(76), Arg(81), Ala(116), Gly(273), and Thr(342) These residues have been specifically replaced by in vitro mutagenesis with Asn, Ser, Val, Pro, and Asn, respectively, as in the Bacillus O16G. The 12 mutant enzymes with single and multiple substitutions were expressed and characterized kinetically. The results showed that the 5-fold mutation virtually abolished the affinity of the enzyme for alpha,alpha'-trehalose, whereas the specificity constant for the hydrolysis of isomaltose, a good substrate for both the SAM1606 enzyme and O16G, remained essentially unchanged upon the mutation. This loss in affinity for trehalose was critically governed by a Gly(273) --> Pro substitution, whose effect was specifically enhanced by the Thr(342) --> Asn substitution in the B-fold and quadruple mutants. These results provide evidence for the differential roles of the amino acid residues in the CR in determining the substrate specificity of the alpha-glucosidase.