Inohara-Ochiai M, Nakayama T, Goto R, Nakao M, Ueda T, Shibano Y
Suntory Research Center, 1-1-1, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618, Japan.
J Biol Chem. 1997 Jan 17;272(3):1601-7. doi: 10.1074/jbc.272.3.1601.
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, Met76, Arg81, Ala116, Gly273, and Thr342. 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 Gly273 --> Pro substitution, whose effect was specifically enhanced by the Thr342 --> Asn substitution in the 5-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.
具有广泛底物特异性的芽孢杆菌属SAM1606 α-葡萄糖苷酶是唯一已知的能够有效水解α,α'-海藻糖的α-葡萄糖苷酶。该酶与嗜热葡萄糖苷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，而后者不能作用于海藻糖。这三种酶在保守区域（CR）内有80%的相同残基，保守区域被认为位于α-淀粉酶家族酶的活性位点附近或活性位点处。为了通过位点特异性诱变确定决定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，并选择了五个靶点在SAM1606 α-葡萄糖苷酶中进行诱变，即Met76、Arg81、Ala116、Gly273和Thr342。如在芽孢杆菌O16G中一样，这些残基分别通过体外诱变被特异性替换为Asn、Ser、Val、Pro和Asn。表达了具有单取代和多取代的12种突变酶并对其进行了动力学表征。结果表明，五重突变几乎消除了该酶对α,α'-海藻糖的亲和力，而对于异麦芽糖（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。对海藻糖亲和力的这种丧失主要由Gly273→Pro取代决定，在五重和四重突变体中，Thr342→Asn取代特异性增强了这种效应。这些结果为CR中的氨基酸残基在决定α-葡萄糖苷酶底物特异性方面的不同作用提供了证据。
