来源于瘤胃菌Ruminococcus sp.的D-阿洛酮糖3-差向异构酶的底物结合位点分析

Substrate-binding site of D-psicose 3-epimerase from Ruminococcus sp.

【目的】研究来源于瘤胃菌Ruminococcus sp.的D-阿洛酮糖3-差向异构酶的底物结合机制。【方法】通过同源模拟和同源序列比对,筛选与其底物结合相关的关键位点,进而通过定点突变构建突变体并对其动力学性质进行研究。【结果】筛选得到关键位点Y6和A109,构建了突变体Y6F、Y6I、A109P及A109L。【结论】Y6既与底物结合又与催化能力相关,其-OH只与底物结合相关,芳香环则与催化能力和结合能力均相关;而A109则只是底物结合的位点。该研究结果为D-阿洛酮糖3-差向异构酶的催化机理研究及分子改造提供了借鉴。

[Objective] The substrate-binding mechanism of D-psicose 3-epimerase from Rumino- coccus sp. has been studied. [Methods] The residues involved in substrate-binding were selected using homology modelling and sequence alignment. Mutants were constructed by site-directed mutagenesis and studied by kinetic analysis. [Results] Two residues, Y6 and A109, were selected. Four mutants, Y6F, Y6I, A109P and A109L were constructed. [Conclusion] Y6 was involved in both catalysis and substrate-binding by the aromatic ring of the amino acid, while the -OH was important for substrate-binding. A109 was an important site that affected binding rather than catalyzing. This research would contribute to the study of catalytic mechanism and molecular modification of D-psicose （D-tagatose） 3-epimerase.