Study on Substrates-Recognition Mechanism of Thermoactinomyces vulgaris R-47 α-Amylase 2 (TVAII) based on X-ray Structures.

普通嗜热放线菌R-47产生两种α-淀粉酶TVAI（637个氨基酸残基，分子量71，OOODa）和TVA II（585个氨基酸残基，分子量67，500 Da），它们不仅能水解淀粉，还能水解环糊精（CD），而其他α-淀粉酶几乎不能水解环糊精。失活突变体TVAIIs的X射线结构（Glu 354->Ala或Asp 325 →Asn）与β-CD复合（E354 A/ β-CD）甲基-β-CD（D325 N/甲基-β-CD）和麦芽六糖（D325 N/G6），表明Phe 286是TVAII识别CD的重要残基，并且G6作为底物的结合模式与在其它α-淀粉酶和α-淀粉酶家族酶中观察到的结合模式非常相似。还对突变体TVAIIs、F286 A、F286 L、F286 Y和F286 W（Phe 286 →Ala、Leu、Tyr、Trp）进行了动力学分析，支持来自X射线结构的结果。在1.6 nm处测定了TVAI的X射线结构，表明TVAI和TVAII在结构上存在一些差异，这与酶的底物特异性和热稳定性有关。

Thermoactinomyces vulgaris R-47 produces two α-amylases TVAI (637 amino acid residues, MW=71,OOODa) and TVAII (585 amino acid residues, MW = 67,500Da), and they can hydrolyze not only starch but also cyclodextrins (CDs), which are scarcely hydrolyzed by other a-amylases. The X-ray structures of inactive mutant TVAIIs (Glu354->Ala or Asp325→Asn) complexed with β-CD (E354A/ β-CD) methyl-β-CD (D325N/methyl-β-CD) and maltohexose (D325N/G6), have been determined, showing that Phe286 is an important residue for TVAII to recognize CDs, and that the binding mode ofG6 as a substrate is very similar to that observed in the other a-amylase and a-amylase family enzymes. Kinetic analyzes of the mutant TVAIIs, F286A, F286L, F286Y and F286W (Phe286→Ala, Leu, Tyr, Trp) have also been done, supporting results from X-ray structures. The X-ray structure of TVAI has been determined at 1.6 Å, showing that there are some differences in the structures between TVAI and TVAII, related to the substrate specificity and thermostability of the enzyme.