SITE SPECIFIC MUTAGENESIS OF H PYLORI CARBONIC ANHYDRASE

幽门螺杆菌碳酸酐酶的位点特异性诱变

• 批准号: 2879847
• 负责人: ROGER S ROWLETT
• 金额: $ 9.07万
• 依托单位: COLGATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-15 至 2002-07-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2879847
• 关键词: Arabidopsis; Helicobacter; X ray crystallography; active sites; carbonate dehydratase; electrophoresis; enzyme activity; enzyme inhibitors; enzyme mechanism; expression cloning; isozymes; nuclear magnetic resonance spectroscopy; protein structure function; recombinant DNA; site directed mutagenesis; stop flow technique

DESCRIPTION: (Verbatim from the Applicant's Abstract) Helicobacter pylori, a human gastric pathogen, codes for two different carbonic anhydrases in its genome, both an alpha and beta form. Because of the importance of carbonic anhydrase in processing cellular carbon dioxide, bicarbonate and protons--all products of the enzyme urease which H. pylori requires for survival in gastric mucosa--it is important to fully understand the structure and function of beta-carbonic anhydrases. One broad goal of the proposed research is to clone, overexpress, and functionally characterize beta-carbonic anhydrase from H. pylori and a closely related carbonic anhydrase from Arabidopsis thaliana. The enzymes will be functionally characterized by stopped-flow spectrophotometry and 13C-NMR exchange kinetics in order to identify the rate-determining step(s) in the mechanism of action, and to partially dissect the mechanism by measuring certain non-rate-determining steps. The enzymes will be structurally characterized by ICP-AES, electrophoresis, and submitted for X-ray crystallographic structure determination. A second broad goal of the proposed research is to further understand the catalytic mechanism and inhibitor binding characteristics of these beta-carbonic anhydrases by engineering site-directed mutants of beta-carbonic anhydrase using recombinant DNA techniques. Mutant enzymes will be overexpressed and functionally and structurally characterized in a manner similar to the wild-type enzymes. Especially interesting mutant enzymes will be submitted for X-ray crystallographic structure determination.

描述：（逐字引用申请人摘要）幽门螺杆菌，a 人类胃病原体，编码两种不同的碳酸酐酶， 基因组，阿尔法和贝塔形式。由于碳的重要性 脱水酶在处理细胞的二氧化碳，碳酸氢盐和质子-所有 H.幽门螺杆菌需要在胃 粘膜-重要的是要充分了解的结构和功能， β-碳酸酐酶。 这项研究的一个广泛目标是克隆，过表达， 功能上表征来自H.幽门螺杆菌和一个密切 来自拟南芥的相关碳酸酐酶。这些酶将 通过停流分光光度法和13 C-NMR进行功能表征 交换动力学，以确定反应中的速率决定步骤。 作用机制，并通过测量部分剖析机制 某些非速率决定步骤。这些酶在结构上 通过ICP-AES、电泳进行表征，并提交X射线 晶体结构测定 拟议研究的第二个广泛目标是进一步了解 催化机理和抑制剂结合特性 通过工程化β-碳酸酐酶的定点突变体 使用重组DNA技术的脱水酶。突变酶将是 过表达，功能和结构特征在于 类似于野生型酶。特别有趣的突变酶将是 提交X射线晶体结构测定。

项目成果

Kinetic characterization of wild-type and proton transfer-impaired variants of beta-carbonic anhydrase from Arabidopsis thaliana.

拟南芥β-碳酸酐酶野生型和质子转移受损变体的动力学特征。

• DOI: 10.1016/s0003-9861(02)00243-6
• 发表时间: 2002
• 期刊: Archives of biochemistry and biophysics
• 影响因子: 3.9
• 作者: Rowlett,RogerS;Tu,Chingkuang;McKay,MelissaM;Preiss,JeffreyR;Loomis,RebeccaJ;Hicks,KatherineA;Marchione,RobbJ;Strong,JacobA;DonovanJr,GeorgeS;Chamberlin,JoyE
• 通讯作者: Chamberlin,JoyE

Age-related onset of obesity corresponds with metabolic dysregulation and altered microglia morphology in mice deficient for Ifitm proteins.

• DOI: 10.1371/journal.pone.0123218
• 发表时间: 2015
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Wee YS;Weis JJ;Gahring LC;Rogers SW;Weis JH
• 通讯作者: Weis JH

Examination of the role of Gln-158 in the mechanism of CO(2) hydration catalyzed by beta-carbonic anhydrase from Arabidopsis thaliana.

检查 Gln-158 在拟南芥 β-碳酸酐酶催化的 CO(2) 水合机制中的作用。

• DOI: 10.1016/j.abb.2004.02.033
• 发表时间: 2004
• 期刊: Archives of biochemistry and biophysics.
• 作者: Rowlett,RogerS;Tu,Chingkuang;Murray,PaulS;Chamberlin,JoyE
• 通讯作者: Chamberlin,JoyE