Effects of essential carbohydrate/aromatic stacking interaction with Tyr100 and Phe259 on substrate binding of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp 1011
Effects of essential carbohydrate/aromatic stacking interaction with Tyr100 and Phe259 on substrate binding of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp 1011
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DOI：
10.1093/jb/mvg215
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发表时间：
2003-12-01
影响因子：
2.7
通讯作者：
Yamane, K
中科院分区：
文献类型：
--
作者：
Haga, K;Kanai, R;Yamane, K
在糖苷水解酶家族13种酶中，酪氨酸残基与催化亚基-1的糖环之间的堆积作用是严格保守的。芽孢杆菌环糊精糖基转移酶(CGTase)中亮氨酸取代Tyr100的研究1011处理显著降低了所有CGTase活性。与Phe183和Phe259在+2亚基糖环上的相邻堆积作用在CGTase中基本上是保守的。F183L/F259L突变体CGTase在转糖基化过程中影响供体底物结合和/或受体结合[Nakamura等人。(1994)生物化学33,9929-9936]。为了阐明-1和+2亚基上的碳水化合物/芳香族堆积相互作用对CGTase底物结合的精确作用，我们分析了野生型(2.0 Angstrom分辨率)、Y100L(2.2 Angstrom分辨率)和F183L/F259L突变体(1.9 Angstrom分辨率)CGTase与抑制剂阿卡波糖复合的X射线结构。精细结构表明，与Y100L突变体结合的阿卡波糖分子从活性中心向Tyr195侧链移动，阿卡波糖与亚基之间的氢键和疏水相互作用显著减弱。F1831L/F259L突变体的假四糖结合位置更接近非还原末端，与采用+2亚位结构变化的野生型阿卡波糖复合体的每个分子相比，分子1上-1至+1亚位和分子2上-2至-1亚位糖苷键的扭转角发生了显著变化。这些结构和生化数据表明，CGTase活性中心的底物结合受催化亚位-1与Tyr100的糖/芳香族堆积相互作用的严重影响，这可能是Tyr100和Phe259通过与+2亚位的堆积相互作用而协同作用的结果。
The stacking interaction between a tyrosine residue and the sugar ring at the catalytic subsite -1 is strictly conserved in the glycoside hydrolase family 13 enzymes. Replacing Tyr100 with leucine in cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. 1011 to prevent stacking significantly decreased all CGTase activities. The adjacent stacking interaction with both Phe183 and Phe259 onto the sugar ring at subsite +2 is essentially conserved among CGTases. F183L/F259L mutant CGTase affects donor substrate binding and/or acceptor binding during transglycosylation [Nakamura et al. (1994) Biochemistry 33, 9929-9936]. To elucidate the precise role of carbohydrate/aromatic stacking interaction at subsites -1 and +2 on the substrate binding of CGTases, we analyzed the X-ray structures of wild-type (2.0 Angstrom resolution), and Y100L (2.2 Angstrom resolution) and F183L/F259L mutant (1.9 Angstrom resolution) CGTases complexed with the inhibitor, acarbose. The refined structures revealed that acarbose molecules bound to the Y100L mutant moved from the active center toward the side chain of Tyr195, and the hydrogen bonding and hydrophobic interaction between acarbose and subsites significantly diminished. The position of pseudo-tetrasaccharide binding in the F1831L/F259L mutant was closer to the non-reducing end, and the torsion angles of glycosidic linkages at subsites -1 to +1 on molecule 1 and subsites -2 to -1 on molecule 2 significantly changed compared with that of each molecule of wild-type-acarbose complex to adopt the structural change of subsite +2. These structural and biochemical data suggest that substrate binding in the active site of CGTase is critically affected by the carbohydrate/aromatic stacking interaction with Tyr100 at the catalytic subsite -1 and that this effect is likely a result of cooperation between Tyr100 and Phe259 through stacking interaction with substrate at subsite +2.