Structure and Mechanisms of PLP Dependent Lyases

PLP 依赖性裂解酶的结构和机制

• 批准号: 7115369
• 负责人: ROBERT STEPHEN PHILLIPS
• 金额: $ 3.78万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7115369
• 关键词: Commonwealth of Independent States; X ray crystallography; active sites; aspartate; chemical kinetics; chemical reaction; decarboxylases; enzyme activity; enzyme mechanism; international cooperation; lyase; methionine; phenols; pyridoxal phosphate; site directed mutagenesis; structural biology; tyrosine

DESCRIPTION (provided by applicant) Enzymes containing pyridoxal-5'-phosphate (PLP) are ubiquitous in biology, performing essential reactions in metabolism of amino acids and amines. These enzymes catalyze a wide variety of reactions, including racemization, transamination, alpha- and beta-decarboxylation, retro-aldol cleavage, beta- and gamma-elimination and substitution. The chemistry of these enzymatic reactions is controlled by the PLP cofactor, and PLP alone has been shown to perform many of these reactions, albeit at very low rates. Therefore, it is the protein environment that confers the reaction specificity and enormous rate accelerations typical of these enzymes. The carbon-carbon lyase, tyrosine phenol-lyase (EC 4.1.99.2, TPL), catalyzes the hydrolytic beta-elimination of L-tyrosine to give phenol and ammonium pyruvate. Methionine-gamma-lyase (EC 4.4.1.11, MGL) catalyzes gamma-elimination reactions of L-methionine to give methylmercaptan and ammonium alpha-ketobutyrate. The main goal of our work is comparative analysis on the molecular level of the principal structure-activity relationships which are characteristic for beta-eliminating (TPL) and gamma-eliminating (MGL) lyases. The most intriguing problem seems to be the transfer of the reaction center from the alpha- to beta-carbon of the substrate. To achieve this goal, a number of experiments will be carried out. Three dimensional structures of intermediates in the reactions of TPL will be determined. Thorough analysis of the catalytic mechanisms of TPL and MGL, depending on the substrate structures, will be performed, using steady state and stopped-flow techniques and multiple isotope effect studies. The enzymes under study are potential drug targets in pathogens. In addition, both TPL and MGL may be useful in cancer therapy. Thus, knowledge of the structure and mechanism of these enzymes may lead to new pharmaceuticals.

描述（由申请人提供） 含有吡哆醛-5 '-磷酸（PLP）的酶在生物学中普遍存在，在氨基酸和胺的代谢中执行必要的反应。这些酶催化各种各样的反应，包括外消旋化、转氨作用、α-和β-脱羧作用、逆向醛醇裂解、β-和γ-消除和取代。这些酶促反应的化学反应由PLP辅因子控制，并且已经显示单独的PLP进行许多这些反应，尽管速率非常低。因此，正是蛋白质环境赋予了这些酶典型的反应特异性和巨大的速率加速。碳-碳裂解酶，酪氨酸苯酚裂解酶（EC 4.1.99.2，TPL）催化L-酪氨酸的水解β-消除以产生苯酚和丙酮酸铵。甲硫氨酸-γ-裂解酶（EC 4.4.1.11，MGL）催化L-甲硫氨酸的γ-消除反应，生成甲硫氨酸和α-酮丁酸铵。我们工作的主要目标是在分子水平上比较分析β-消除（TPL）和γ-消除（MGL）裂解酶的主要结构-活性关系。最有趣的问题似乎是反应中心从底物的α-碳转移到β-碳。为了实现这一目标，将进行一些实验。确定了TPL反应中间体的三维结构。TPL和MGL的催化机制的彻底分析，取决于基板结构，将进行，使用稳态和停流技术和多种同位素效应的研究。正在研究的酶是病原体中潜在的药物靶点。此外，TPL和MGL都可用于癌症治疗。因此，了解这些酶的结构和机制可能会导致新的药物。

项目成果

Insights into the catalytic mechanism of tyrosine phenol-lyase from X-ray structures of quinonoid intermediates.

• DOI: 10.1074/jbc.m802061200
• 发表时间: 2008-10-24
• 期刊: The Journal of biological chemistry
• 作者: Milić D;Demidkina TV;Faleev NG;Matković-Calogović D;Antson AA
• 通讯作者: Antson AA

Structures of apo- and holo-tyrosine phenol-lyase reveal a catalytically critical closed conformation and suggest a mechanism for activation by K+ ions.

• DOI: 10.1021/bi0601858
• 发表时间: 2006-06-20
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Milić D;Matković-Calogović D;Demidkina TV;Kulikova VV;Sinitzina NI;Antson AA
• 通讯作者: Antson AA

High-resolution structure of methionine gamma-lyase from Citrobacter freundii.

弗氏柠檬酸杆菌蛋氨酸 γ-裂合酶的高分辨率结构。

• DOI: 10.1107/s0907444907065390
• 发表时间: 2008
• 期刊: Acta crystallographica. Section D, Biological crystallography
• 作者: Nikulin,Alexei;Revtovich,Svetlana;Morozova,Elena;Nevskaya,Natalia;Nikonov,Stanislav;Garber,Maria;Demidkina,Tatyana
• 通讯作者: Demidkina,Tatyana

Kinetic and spectral parameters of interaction of Citrobacter freundii methionine γ-lyase with amino acids.

弗氏柠檬酸杆菌蛋氨酸γ-裂解酶与氨基酸相互作用的动力学和光谱参数。

• DOI: 10.1134/s0006297910100093
• 发表时间: 2010
• 期刊: Biochemistry. Biokhimiia
• 作者: Morozova,EA;Bazhulina,NP;Anufrieva,NV;Mamaeva,DV;Tkachev,YV;Streltsov,SA;Timofeev,VP;Faleev,NG;Demidkina,TV
• 通讯作者: Demidkina,TV