Okuyama Masayuki, Kaneko Akira, Mori Haruhide, Chiba Seiya, Kimura Atsuo
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-9 Nishi-9, Sapporo 060-8589, Japan.
FEBS Lett. 2006 May 15;580(11):2707-11. doi: 10.1016/j.febslet.2006.04.025. Epub 2006 Apr 19.
Escherichia coli YicI, a member of glycoside hydrolase family (GH) 31, is an alpha-xylosidase, although its amino-acid sequence displays approximately 30% identity with alpha-glucosidases. By comparing the amino-acid sequence of GH 31 enzymes and through structural comparison of the (beta/alpha)(8) barrels of GH 27 and GH 31 enzymes, the amino acids Phe277, Cys307, Phe308, Trp345, Lys414, and beta-->alpha loop 1 of (beta/alpha)(8) barrel of YicI have been identified as elements that might be important for YicI substrate specificity. In attempt to convert YicI into an alpha-glucosidase these elements have been targeted by site-directed mutagenesis. Two mutated YicI, short loop1-enzyme and C307I/F308D, showed higher alpha-glucosidase activity than wild-type YicI. C307I/F308D, which lost alpha-xylosidase activity, was converted into alpha-glucosidase.
大肠杆菌YicI是糖苷水解酶家族（GH）31的成员，是一种α-木糖苷酶，尽管其氨基酸序列与α-葡萄糖苷酶的序列相似度约为30%。通过比较GH 31酶的氨基酸序列，并对GH 27和GH 31酶的（β/α）8桶状结构进行结构比较，已确定YicI的（β/α）8桶状结构中的氨基酸Phe277、Cys307、Phe308、Trp345、Lys414以及β→α环1可能是影响YicI底物特异性的重要元件。为了将YicI转化为α-葡萄糖苷酶，已通过定点诱变对这些元件进行了改造。两种突变的YicI，即短环1酶和C307I/F308D，显示出比野生型YicI更高的α-葡萄糖苷酶活性。失去α-木糖苷酶活性的C307I/F308D已转化为α-葡萄糖苷酶。
