来源于Pseudomonas saccharophila STB07的麦芽四糖生成酶的结构与催化机制分析
首发时间:2019-02-08
摘要:本文以来源于Pseudomonas saccharophila STB07的麦芽四糖酶(MFAps酶)为研究对象,通过悬滴法与分子置换得到了高分辨率的MFAps酶单一结构及其与底物复合结构,重点分析了氨基酸与底物的构象和相互作用。结果显示,MFAps酶的活性中心位于(β/α)8桶状结构的表面,麦芽四糖(G4)结合在MFAps酶的-4~-1亚位点,麦芽七糖类似物(pG7)结合在-4~+3亚位点。在-4位点,Gly158、Glu160与Ser161可能与底物非还原末端的识别有关,协同-3位点的Trp66将底物引导并定位至活性中心;-1与+1位点的3个催化残基Asp193、Glu219和Asp294通过侧链构象的变化减小底物与酶结合的阻力并完成催化过程,同时能够与-1位点葡萄糖残基形成疏水堆积的Tyr78也可能对酶的催化活力有一定影响;在+2与+3位点,主要依靠Trp221和Trp308提供的疏水堆积与水分子提供的氢键将底物稳定在酶的表面,加速反应的进行。研究发现,催化残基向底物非还原末端的4个亚位点对产物特异性起到了决定作用,并且氨基酸与葡萄糖残基之间的强相互作用对底物催化过程有重要影响。
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The analysis of structure and catalytic mechanism of maltotetraose-forming amylase derived from Pseudomonas saccharophila STB07
Abstract:This study focused on maltotetraose-forming amylase (MFAps) derived from Pseudomonas saccharophila STB07. To elucidate the interaction between active residues and substrates as well as their conformation emphatically, high-quality protein crystals of MFAps and its complex with substrates were obtained by hanging drop method and resolved by molecular replacement method. Results showed that MFAps has a common (β/α)8 barrel structure with a cavity including catalytic sites. Maltotetraose (G4) and pseudo-maltoheptaose (pG7) occupy subsites -4~-1 and subsites -4~+3 in their complexes with MFAps respectively. At subsite -4, Gly158, Glu160 and Ser161 may be involved in the recognition of the non-reducing end of the substrate, while Trp66 at subsite -3 site directs and localizes the substrate to the active center in coThe analysis of structureThe analysis of structure and catalytic mechanism of maltotetraose-forming amylase derived from Pseudomonas saccharophila STB07 and catalytic mechanism of maltotetraose-forming amylase derived from Pseudomonas saccharophila STB07ordination; side chains of three catalytic residues, Asp193, Glu219 and Asp294, are induced to conformational changes leading to steric hindrance reduced when substrates binding at subsites -1 and +1; moreover, the indole moieties of Trp221 and Trp308 are located at subsites +2 and +3, right above glucosyl residues, stabilizing the substrates and accelerating the reaction through hydrophobic stacks which combine with hydrogen bonds provided by water. In general, this study revealed that subsites -4~-1 are presumed to decide its product specificity and the strong interactions of active residues with glucosyl residues are responsible to catalytic reactions.
Keywords: maltotetraose-forming amylase structure interaction catalytic mechanism
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来源于Pseudomonas saccharophila STB07的麦芽四糖生成酶的结构与催化机制分析
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