Studies of Isoprenoid Biosynthesis

类异戊二烯生物合成的研究

基本信息

批准号：
8645630
负责人：
CHARLES DALE POULTER
金额：
$ 39万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
1979
资助国家：
美国
起止时间：
1979-04-01 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8645630
关键词：
Active Sites Amines Amino Acids Anabolism Anti-Bacterial Agents Antibiotics Antifungal Agents Antiparasitic Agents Archaea Area Artemisinins Bacteria Biochemical Biological Factors Blood Vessels Carbon Carboxy-Lyases Catalysis Cell division Cells Chemicals Cholesterol Collection Complement Complex Coronary heart disease Custom Decarboxylation Development Diphosphates Disease Drug Targeting Elements Engineering Enzymatic Biochemistry Enzyme Gene Enzyme Inhibitor Drugs Enzyme Inhibitors Enzymes Escherichia coli Eukaryota Family Flavin Mononucleotide Flavins Fluorescence Fosamax Genes Genetic Glycolysis Goals Grant Growth Health Human Individual Ions Iron Isomerase Isoprenoid Phosphates Kinetics Knock-out Laboratories Ligands Link Malaria Malignant Neoplasms Measurement Measures Mediating Metalloproteins Metals Molecular Biology Mutagenesis Myopathy Nature Nomenclature Open Reading Frames Organic Chemistry Organism Osteoporosis Paclitaxel Pathway interactions Pattern Pharmaceutical Preparations Pharmacologic Substance Phosphorylation Phosphotransferases Procedures Process Production Protein Isoforms Proteins Protons Publishing Reaction Recombinants Recording of previous events Regulation Research Research Support Respiration Roentgen Rays Role Route Serum Site Site-Directed Mutagenesis Staging Steroid biosynthesis Stroke Structure Sulfhydryl Compounds Sulfur Technology Vision Work Yeasts adduct analog analytical tool artemisinine atorvastatin base chemical binding cofactor design directed evolution drug candidate drug development enzyme pathway enzyme substrate expression vector farnesyl pyrophosphate fatty acid metabolism fosmidomycin gene synthesis genetic linkage analysis high throughput screening human disease hydroxyl group in vivo inhibitor/antagonist inorganic phosphate isopentenyl pyrophosphate isoprenoid mevalonate microorganism mutant new technology novel pathogenic bacteria phosphomevalonate research study small molecule structural biology

项目摘要

DESCRIPTION (provided by applicant): Isoprenoid compounds comprise the largest family of small molecules in nature. Many are essential for cells to survive and elements of the isoprenoid biosynthetic pathway are found in all organisms. Several isoprenoid biosynthetic enzymes are targets for drugs. Lipitor, the most widely prescribed drug, targets an enzyme early in the pathway to lower serum cholesterol. Fosamax, used to treat osteoporosis, targets a chain elongation enzyme in the middle of the pathway. The antifungal drug Lamisil targets the later stages of fungal steroid biosynthesis. Differences between the isoprenoid biosynthetic pathways and enzymes in humans and microorganisms present new attractive targets for drug development. In addition, products of the pathway have a long history as drugs, including more recent examples such as taxol (cancer) and artemisinin (malaria). This project supports research to elucidate new biosynthetic routes to isoprenoid compounds and structural and mechanistic studies of enzymes in the pathways from where the routes diverge from fatty acid metabolism and glycolysis up to the synthesis of the two fundamental five-carbon building blocks of isoprenoid molecules, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). The work incorporates a combination of genetics, molecular biology, mechanistic enzymology, structural biology, and synthetic organic chemistry in this effort. The PI's laboratory is a unique site for this project. He and his coworkers have expertise specifically related to the isoprenoid pathway in the diverse areas of research outlined above. His laboratory has a large collection of bacterial and fungal clones bearing knockouts or additions of appropriate biosynthetic enzymes, expression vectors for production of purified enzymes, substrates/alternate substrates/inhibitors, and the analytical tools required for identification of new metabolites and for structural and mechanistic studies. Three interrelated areas of research are proposed for the coming grant period. (1) All available evidence indicates that most, if not all, Archaea synthesize isopentenyl diphosphate from mevalonate differently that eukaryotes. Specifically, mevalonate phosphate is first converted to isopentenyl phosphate (IP) and then to the diphosphate. The gene for synthesis of IP is missing. Identification of the gene and verification of its function will be undertaken. Related work on X-ray structure-function and mutagenesis studies of IP kinase will be conducted, including the development of mutants capable of efficiently synthesizing longer chain diphosphates from the corresponding phosphates. (2) Isoforms of IPP isomerase (IDI1 and IDI2) convert IPP to DMAPP by a proton-initiated process. NMR, X-ray, UV, and fluorescence experiments will be conducted to identify the amino acids/cofactors involved in the proton transfer reactions, including the unusual role of reduced flavin in the proton transfer reactions of IDI2. (3) Amine and thiol-containing analogues of methylerythritol phosphate and hydroxydimethylallyl diphosphate will be synthesized and examined as mechanism-based inhibitors of the iron-sulfur cluster enzymes the methylerythritol phosphate pathway.

描述（由申请人提供）：类异型化合物包括自然界中最大的小分子家族。许多对于细胞生存至关重要，在所有生物体中都发现了类异丙生素生物合成途径的元素。几种类异丙生素生物合成酶是药物的靶标。 LIPITOR是规定的最广泛的药物，它靶向低血清胆固醇的途径早期。用于治疗骨质疏松症的Fosamax靶向途径中间的链伸长酶。抗真菌药物的lamisil靶向真菌类固醇生物合成的后期。类人和微生物的异急动物生物合成途径和酶之间的差异提出了药物开发的新诱人靶标。此外，该途径的产物作为药物的历史悠久，包括紫杉醇（癌症）和青蒿素（疟疾）等最近的例子。 This project supports research to elucidate new biosynthetic routes to isoprenoid compounds and structural and mechanistic studies of enzymes in the pathways from where the routes diverge from fatty acid metabolism and glycolysis up to the synthesis of the two fundamental five-carbon building blocks of isoprenoid molecules, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate （DMAPP）。这项工作结合了遗传学，分子生物学，机械酶学，结构生物学和合成有机化学的组合。 PI的实验室是该项目的独特站点。他和他的同事在上面概述的不同研究领域中具有与类吸引途径特别相关的专业知识。他的实验室拥有大量的细菌和真菌克隆，这些克隆具有敲除或添加适当的生物合成酶，用于生产纯化酶的表达向量，底物/替代基底物/抑制剂，以及用于鉴定新代谢物的分析工具以及用于结构和机械性研究所需的分析工具。在未来的赠款期间提出了三个相互关联的研究领域。（1）所有可用的证据表明，大多数（如果不是全部）的古细菌合成二磷酸二磷酸二磷酸二磷酸二磷酸酯的含量与真核生物不同。具体而言，首先将甲戊酸磷酸盐转化为等烯基磷酸盐（IP），然后转化为双磷酸盐。缺少用于合成IP的基因。将进行识别基因和其功能验证。将进行有关IP激酶的X射线结构功能和诱变研究的相关工作，包括开发能够有效合成相应磷酸盐的较长链二磷酸的突变体。（2）IPP异构酶（IDI1和IDI2）的同工型通过质子引发的过程将IPP转换为DMAPP。将进行NMR，X射线，UV和荧光实验，以鉴定参与质子转移反应的氨基酸/辅因子，包括黄素在IDI2的质子转移反应中的异常作用。（3）将合成甲基疫醇磷酸盐和羟基二甲基二磷酸的胺和含硫醇的类似物，并将其作为基于机制的铁硫簇酶的基于机制的抑制剂检查。

项目成果

期刊论文数量（57）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Crystal structure of type 2 isopentenyl diphosphate isomerase from Thermus thermophilus in complex with inorganic pyrophosphate.

DOI：
10.1021/bi801159x
发表时间：
2008-09-02
期刊：
BIOCHEMISTRY
影响因子：
2.9
作者：
de Ruyck, Jerome;Pouyez, Jenny;Rothman, Steven C.;Poulter, Dale;Wouters, Johan
通讯作者：
Wouters, Johan

Synthesis of 1-Deoxy-D-xylulose and 1-Deoxy-D-xylulose-5-phosphate.

1-脱氧-D-木酮糖和1-脱氧-D-木酮糖-5-磷酸的合成。