微囊藻毒素降解酶MlrA的结构功能分析

Structural and functional analysis of MlrA from microcystin-degrading bacteria

MlrA(亦称microcystinase)是微囊藻毒素(microcystins,MCs)细菌降解途径中负责催化起始反应的关键蛋白酶,其结构特征与底物水解机制尚未明确。使用折叠识别法构建MlrA分子模型,通过分子对接和定点突变分析了酶-底物的结合方式与相互作用,结合蛋白重组表达对酶活性影响机制等进行了探究。结果表明MlrA是定位于细菌细胞质膜的整合膜蛋白,主要由8个跨膜α-螺旋(TM1~8)组成,功能结构域ABI(TM4~7)形成向周质空间开放的底物反应空腔。MlrA催化残基(E172、H205、H260和N264)位于膜内,其侧链投射至反应腔内部。微囊藻毒素LR(MC-LR)采用β-发夹构型与酶结合并将易裂键暴露于水分子附近,其水解机制为E172和H205通过一般碱催化将水分子去质子化激活,对Adda-Arg肽键羰基碳进行亲核攻击;接着H260和N264构成氧阴离子穴以稳定过渡态氧阴离子;最后H205或E172催化胺离去基团发生质子化,使四面体氧阴离子中间体崩解。此外,MlrA不是金属蛋白酶,无法与金属离子(Ⅱ)配位结合,菲咯啉类化合物使酶分子发生非特异性解折叠而失活,EDTA对底物结合位点具有竞争作用。本研究揭示了MlrA的属性与水解机制,为进一步探索MCs微生物降解机理提供一定参考依据。

The critical enzyme responsible for catalyzing the first step in the microcystin(MC)biodegradation pathway identified in bacterial strains is referred to as MlrA(also known as microcystinase).Its structural characteristics and substrate hydrolysis mechanism are not yet clear.In this paper,a molecular structure model of MlrA was established using a fold recognition method.Docking and site directed mutagenesis approach was employed to determine the binding modes and interaction network of enzyme-ligand complex.Factors affecting the catalytic activity of the enzyme were also elucidated by in vitro enzyme assays.The results show that MlrA is an integral membrane protein localized at the plasma membrane,its structure comprising mainly of eight transmembraneα-helices(TM1—8),in which the conserved ABI domain(TM4—7)forms a conical cavity with a large volume and opens to the periplasmic space.The catalytic residues(E172,H205,H260 and N264)are located into the membrane that projects their side chains into the cavity for catalysis.The MC-LR adopts aβ-hairpin structure within a cavity to bind to MlrA,which then positions the scissile bond adjacent to a water molecule.Thus,a complete proteolytic mechanism of the enzyme was proposed.First,E172 and H205 general base-catalyzed deprotonation of a water molecule for nucleophilic attack on the Adda-Arg peptide bond.Then,H260 and N264,which form an oxyanion hole,stabilize the oxyanion transition state by hydrogen bonding.Finally,protonation of the amine leaving group of Adda-Arg peptide bond could be catalyzed by either H205 or E172 to allow collapse of the intermediate.Furthermore,this enzyme does not contain a metal-binding site(Ⅱ),its concentration-dependent inactivation by phenanthroline results from non-specific protein unfolding,EDTA competes for the binding site of the enzyme by developing an interaction network within the active site similar to that of the substrate.These findings suggest that MlrA is not a metalloproteinase.This study revealed the properties and hydrolysis mechanism of MlrA,and provided a reference for further exploration of the microbial degradation mechanism of MCs.