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 5'-磷酸吡哆醛 (PLP) 的酶在生物学中无处不在，在氨基酸和胺的代谢中执行重要反应。这些酶催化多种反应，包括外消旋化、转氨基作用、α-和β-脱羧、逆羟醛裂解、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，并且已证明仅 PLP 即可执行许多此类反应，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，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，产生甲硫醇和 α-酮丁酸铵。我们工作的主要目标是在分子水平上对β-消除（TPL）和γ-消除（MGL）裂解酶的主要结构-活性关系进行比较分析。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，将进行大量实验。 TPL反应中中间体的三维结构将被确定。将使用稳态和停流技术以及多种同位素效应研究，根据底物结构对 TPL 和 MGL 的催化机制进行彻底分析。正在研究的酶是病原体的潜在药物靶点。此外，TPL 和 MGL 都可能有助于癌症治疗。因此，了解这些酶的结构和机制可能会带来新的药物。

项目成果

Methionine gamma-lyase: mechanistic deductions from the kinetic pH-effects. The role of the ionic state of a substrate in the enzymatic activity.

蛋氨酸 γ-裂解酶：从动力学 pH 效应中进行机械推论。

• 发表时间: 2009-10
• 作者: Faleev, Nicolai G;Alferov, Kirill V;Tsvetikova, Marina A;Morozova, Elena A;Revtovich, Svetlana V;Khurs, Elena N;Vorob'ev, Mikhail M;Phillips, Robert S;Demidkina, Tatyana V;Khomutov, Radii M
• 通讯作者: Khomutov, Radii M

A gene encoding L-methionine gamma-lyase is present in Enterobacteriaceae family genomes: identification and characterization of Citrobacter freundii L-methionine gamma-lyase.

肠杆菌科基因组中存在编码 L-蛋氨酸 γ-裂解酶的基因：弗氏柠檬酸杆菌 L-蛋氨酸 γ-裂解酶的鉴定和表征。

• 发表时间: 2005-06
• 作者: Manukhov, Ilya V;Mamaeva, Daria V;Rastorguev, Sergei M;Faleev, Nicolai G;Morozova, Elena A;Demidkina, Tatyana V;Zavilgelsky, Gennadii B
• 通讯作者: Zavilgelsky, Gennadii B

Structure of Citrobacter freundii L-methionine gamma-lyase.

弗氏柠檬酸杆菌 L-甲硫氨酸 γ-裂解酶的结构。

• 发表时间: 2005-06-01
• 作者: Mamaeva, D V;Morozova, E A;Nikulin, A D;Revtovich, S V;Nikonov, S V;Garber, M B;Demidkina, T V
• 通讯作者: Demidkina, T V

L-methionine gamma-lyase from Citrobacter freundii: cloning of the gene and kinetic parameters of the enzyme.

来自弗氏柠檬酸杆菌的 L-蛋氨酸 γ-裂合酶：基因克隆和酶的动力学参数。

• 发表时间: 2006-04
• 作者: Manukhov, I V;Mamaeva, D V;Morozova, E A;Rastorguev, S M;Faleev, N G;Demidkina, T V;Zavilgelsky, G B
• 通讯作者: Zavilgelsky, G B

Exploring methionine γ-lyase structure-function relationship via microspectrophotometry and X-ray crystallography.

• DOI: 10.1016/j.bbapap.2010.06.017
• 发表时间: 2011-06-01
• 期刊: Biochimica et biophysica acta
• 作者: L. Ronda;N. P. Bazhulina;E. Morozova;S. Revtovich;V. Chekhov;A. Nikulin;T. Demidkina;A. Mozzarelli
• 通讯作者: A. Mozzarelli