Enzymatic Mechanisms of Two Pyrimidine Decarboxylases

两种嘧啶脱羧酶的酶机制

基本信息

批准号：
6748224
负责人：
JEFFREY A SMILEY
金额：
$ 0.45万
依托单位：
YOUNGSTOWN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-09-01 至 2005-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Pyrimidine metabolic enzymes have long been recognized as targets in which drug intervention can be effective in slowing the growth of infectious microorganisms or rapidly proliferating cells. In two pyrimidine metabolic pathways -the de novo pathway and the thymidine salvage pathway, a rare pathway apparently present only in some fungi - the final pyrimidine base modification step to yield the uracil ring is a decarboxylation. The two decarboxylases that catalyze these reactions are OMP decarboxylase (ODCase) and iso-orotate decarboxylase (IDCase), respectively. The long-term objective of this project is to contribute to a complete elucidation of the mechanisms of these two decarboxylases, allowing a rational approach to the feasibility of these enzymes as inhibitor sites in pyrimidine metabolism. The specific aims for the study of ODCase will be to verify whether or not the active site lysine residue (Lys93 of the yeast enzyme), identified recently in crystal structures of four ODCases from different organisms, contacts O2 of the substrate, as originally postulated. This will be addressed using spectroscopic studies of ODCase in complex with various inhibitors: 1) 1H NMR spectroscopy of engineered ODCase with 15N specifically labeled at the active site amino group; 2) NMR spectroscopy of ODCase complexed with inhibitors with 15N, 13C1' and 1H1' and 3) Raman difference spectroscopy of ODCase/inhibitor complexes, to determine whether or not O2 of the pyrimidine nucleotide inhibitor is polarized by hydrogen bonding to the enzyme. The first specific aim for the study of IDCase will be to develop an effective affinity chromatography purification method based on the observed tight binding of the enzyme to 5-nitrouracil. Upon adequate enzyme purification, kinetic measurements will be determined for substrate and inhibitor binding, which may include inhibition by pharmacologically significant 5-fluorinated pyrimidines. Adequate enzyme purification will also allow examination of the mode of binding of 5-nitrouracil using NMR spectroscopy of the IDCase/[13C5] 5-nitrouracil complex. Finally, adequate protein purification will allow N-terminal amino acid sequencing, leading to the design of oligonucleotide probes that will be used in an attempt to locate the IDCase gene in a cDNA library of genes from Rhodotorula glutinis.

描述（由申请人提供）：长期以来，嘧啶代谢酶一直被认为是药物干预可有效治疗的靶标。减缓传染性微生物的生长或快速增殖的细胞。在两种嘧啶代谢途径中——从头途径和胸苷补救途径，一种罕见的途径，显然仅存在于某些真菌中 - 产生尿嘧啶环的最终嘧啶碱基修饰步骤是脱羧。催化这些反应的两种脱羧酶是 OMP 分别是脱羧酶（ODCase）和异乳清酸脱羧酶（IDCase）。该项目的长期目标是为完整的阐明这两种脱羧酶的机制，允许合理的探讨这些酶作为嘧啶抑制剂位点的可行性代谢。 ODCase 研究的具体目的是验证是否活性位点赖氨酸残基（酵母酶的 Lys93），最近在来自不同生物体的四种 ODCases 的晶体结构，接触 O2 基质，如最初假设的那样。这将通过光谱来解决 ODCase 与各种抑制剂复合物的研究：1) 1H NMR 谱工程化 ODCase，在活性位点氨基上专门标记 15N； 2) 与 15N、13C1' 和 15N、13C1' 抑制剂复合的 ODCase 的 NMR 谱 1H1' 和 3) ODCase/抑制剂复合物的拉曼差异光谱，以确定嘧啶核苷酸抑制剂的O2是否极化通过氢键与酶结合。 IDCase 研究的第一个具体目标是开发一种有效的基于观察到的紧密结合的亲和层析纯化方法酶转化为5-硝基尿嘧啶。经过充分的酶纯化，动力学将确定底物和抑制剂结合的测量值，这可能包括具有药理学意义的5-氟化嘧啶的抑制作用。充分的酶纯化还可以检查结合模式使用 IDCase/[13C5] 5-硝基尿嘧啶的 NMR 光谱分析 5-硝基尿嘧啶复杂的。最后，充分的蛋白质纯化将使 N 端氨基酸测序，从而设计出寡核苷酸探针用于尝试在 cDNA 基因文库中定位 IDCase 基因粘红酵母。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A substantial oxygen isotope effect at O2 in the OMP decarboxylase reaction: mechanistic implications.

OMP 脱羧酶反应中 O2 的显着氧同位素效应：机制意义。

DOI：
发表时间：
2008-12-21
期刊：
影响因子：
3.2
作者：
Wepukhulu, Wickliffe O;Smiley, Vanessa L;Vemulapalli, Bhargavi;Smiley, Jeffrey A;Phillips, Linda M;Lee, Jeehiun K
通讯作者：
Lee, Jeehiun K

Hydrogen isotope tracing in the reaction of orotidine-5'-monophosphate decarboxylase.

乳清苷-5-单磷酸脱羧酶反应中的氢同位素示踪。

DOI：
发表时间：
2003-04-15
期刊：
影响因子：
3.9
作者：
Smiley, Jeffrey A;DelFraino, Brian J;Simpson, Beth A
通讯作者：
Simpson, Beth A

A novel enzyme complex of orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase in human malaria parasite Plasmodium falciparum: physical association, kinetics, and inhibition characterization.

人疟原虫恶性疟原虫中乳清酸磷酸核糖基转移酶和乳清苷 5-单磷酸脱羧酶的新型酶复合物：物理关联、动力学和抑制特性。

DOI：
发表时间：
2005-02-08
期刊：
影响因子：
0
作者：
Krungkrai, Sudaratana R;DelFraino, Brian J;Smiley, Jeffrey A;Prapunwattana, Phisit;Mitamura, Toshihide;Horii, Toshihiro;Krungkrai, Jerapan
通讯作者：
Krungkrai, Jerapan

Genes of the thymidine salvage pathway: thymine-7-hydroxylase from a Rhodotorula glutinis cDNA library and iso-orotate decarboxylase from Neurospora crassa.

胸苷补救途径的基因：来自粘红酵母 cDNA 文库的胸腺嘧啶-7-羟化酶和来自粗糙脉孢菌的异乳清酸脱羧酶。