Real-time investigation of mannosyltransferase function of a Xylella fastidiosa recombinant GumH protein using QCM-D
Real-time investigation of mannosyltransferase function of a Xylella fastidiosa recombinant GumH protein using QCM-D
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DOI：
10.1016/j.bbrc.2011.04.062
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发表时间：
2011-05-20
影响因子：
3.1
通讯作者：
Faria, Ronaldo C.
中科院分区：
文献类型：
--
作者：
Alves, Claudia A.;Pedroso, Mariele M.;Faria, Ronaldo C.
苛养木杆菌是一种革兰氏阴性细菌，在重要的经济作物，包括葡萄藤，咖啡和柑橘类水果中引起严重疾病。X. fastidiosa定殖于受感染植物的木质部导管，从而阻断水和养分的运输。对X. fastidiosa的研究揭示了一个操纵子，该操纵子含有9个基因，可能参与合成一种名为fastidian gum的胞外多糖（EPS），这种胞外多糖可能与该细菌的致病性有关。α-1，3-甘露糖基转移酶（GumH）酶来自X. fastidiosa参与fastidian胶的产生。GumH负责在EPS的组装和生物合成期间将甘露糖从鸟苷二磷酸甘露糖（GDP-man）转移到纤维二糖-焦磷酸-聚戊烯醇载体脂质（CPP-Lip）。在这项工作中，一种实时检测重组GumH酶活性的方法，成功地使用石英晶体微天平与耗散监测（QCM-D）。QCM-D传感器策略性地用CPP-Lip通过使用固体支撑的脂质双层进行修饰，所述脂质双层利用1-十一烷醇的自组装单层。在GDP-man和GumH酶存在下监测实时CPP-Lip QCM-D换能器显示质量增加，表明甘露糖的转移。通过开发用于测定酶促反应产生的GDP的高效液相色谱（LC）方法验证了甘露糖基转移酶功能的实时QCM-D测定。LC结果证实了重组GumH蛋白的活性，它是参与X. fastidiosa的酶促特征。(C)2011 Elsevier Inc. All rights reserved.
Xylella fastidiosa is a gram-negative bacterium that causes serious diseases in economically important crops, including grapevine, coffee, and citrus fruits. X. fastidiosa colonizes the xylem vessels of the infected plants, thereby blocking water and nutrient transport. The genome sequence of X. fastidiosa has revealed an operon containing nine genes possibly involved in the synthesis of an exopolisaccharide (EPS) named fastidian gum that can be related with the pathogenicity of this bacterium. The alpha-1,3-mannosyltransferase (GumH) enzyme from X. fastidiosa is involved in fastidian gum production. GumH is responsible for the transfer of mannose from guanosine diphosphate mannose (GDP-man) to the cellobiose-pyrophosphate-polyprenol carrier lipid (CPP-Lip) during the assembly and biosynthesis of EPS. In this work, a method for real-time detection of recombinant GumH enzymatic activity was successfully developed using a Quartz Crystal Microbalance with dissipation monitoring (QCM-D). The QCM-D transducer was strategically modified with CPP-Lip by using a solid-supported lipid bilayer that makes use of a self-assembled monolayer of 1-undecanethiol. Monitoring the real-time CPP-Lip QCM-D transducer in the presence of GDP-man and GumH enzyme shows a mass increase, indicating the transfer of mannose. The real-time QCM-D determination of mannosyltransferase function was validated by a High Performance Liquid Chromatography (LC) method developed for determination of GDP produced by enzymatic reaction. LC results confirmed the activity of recombinant GumH protein, which is the first enzyme involved in the biosynthesis of the EPS from X. fastidiosa enzymatically characterized. (C) 2011 Elsevier Inc. All rights reserved.