Dissecting the substrate specificity of acyl-CoA carboxylase

剖析酰基辅酶A羧化酶的底物特异性

基本信息

批准号：
7790023
负责人：
Shiou-Chuan Tsai
金额：
$ 6.97万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-01 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7790023
关键词：
Acetyl Coenzyme A Acetyl-CoA Carboxylase Active Sites Acyl Coenzyme A Affect Anabolism Antibiotics Bacteria Binding Biochemistry Biological Factors Biomimetics Bioterrorism Biotin Chemicals Cholesterol Commit Communities Data Deficiency Diseases Development Environment General Population Goals Herbicides Human Investigation Knowledge Link Metabolic Metabolic Diseases Molecular Multienzyme Complexes Mutagenesis Mutate Mutation Outcome Pharmaceutical Preparations Pharmacologic Substance Production Proteins Public Health Reaction Research Running Screening procedure Specificity Streptomyces Streptomyces coelicolor Structure Substrate Specificity Testing Therapeutic Time Training Virus anticancer activity base biological systems carboxylation design fatty acid biosynthesis genetic analysis graduate student inhibitor/antagonist innovation ketotic hyperglycinemia methylmalonyl-CoA decarboxylase methylmalonyl-coenzyme A molecular recognition mutant novel therapeutics propionyl-coenzyme A public health relevance structural biology

项目摘要

DESCRIPTION (provided by applicant): Acyl-coenzyme A carboxylases (ACCases), such as acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC), catalyze the carboxylation of acetyl- and propionyl-CoA to provide malonyl- and methylmalonyl-CoA, respectively. This carboxylation reaction is ubiquitously important in biological systems, because it commits acetyl-CoA and propionyl-CoA to the biosyntheses of fatty acids, polyketides and Kreb cycle intermediates. While there is a well-developed body of knowledge on the genetic analysis, mechanistic and biomimetic studies of ACCases, the development of ACCase-related therapeutics has been severely hampered by the lack of molecular information on how ACCases recognize their corresponding substrates or inhibitors. Our long-term goal is to generate ACCase-based therapeutics and to screen for their pharmaceutical activities. The objective of this particular application, which is the next step toward our long-term goal, is to determine the molecular basis of substrate specificity of ACC and PCC from Streptomyces coelicolor. The S. coelicolor ACCases provide extender units to the biosynthesis of polyketides, a class of natural products that include many antibiotic, anticancer and cholesterol-lowering pharmaceuticals. Mutant ACCases can potentially provide new building blocks to polyketide biosynthesis, so that new polyketides with altered extender units can be biosynthesized. These new polyketides, with the antibiotic chemical templates, will be excellent drug leads to be screened against bioterrorism targets of bacteria and viruses. The central hypothesis is that it should be possible to use mutagenesis to change the substrate specificity of ACCase for the purpose of generating new extender units for polyketide biosynthesis. We base the hypothesis on the observation that 1) ACCase subunits have distinct specificity for different substrate and inhibitors; 2) our preliminary data on the structures and functions of the ACCase 2-subunits (AccB and PccB) have identified specific residues that are responsible for molecular recognition. If the hypothesis is true, mutant ACCases will produce new substituted malonyl-CoAs, which can serve as new extender units for polyketide biosynthesis. We will pursue two specific aims: AIM 1. SOLVE COCRYSTAL STRUCTURES OF ACCB AND PCCB: 1.1. Solve protein-substrate cocrystal structures. 1.2. Solve protein-regulator cocrystal structures. AIM 2. MAKE ACTIVE SITE MUTANTS OF ACCB AND PCCB: 2.1. Systematically mutate residue 422. 2.2. Mutate residues in the acyl-CoA binding pocket. 2.3. Mutate residues in the biotin binding pocket. Once we identify the residues that can be mutated to change the specificity of ACCase, it will become possible to generate new, substituted malonyl-CoAs that can serve as new extender units for polyketide biosynthesis. This innovative approach has not been undertaken before. Because of our research focus and the complementary expertise, our research environment is especially conductive to successful completion of the proposed investigations on ACCases. The research proposed in this application is significant, because its outcome allows us to dissect the molecular features that are responsible for substrate specificity of ACCases. In the long run, the result from this proposal will have a significant positive impact on the development of new antibiotics that are either ACCase inhibitors (for blocking fatty acid biosynthesis of bacteria) or have new extender units (for the biosynthesis of new polyketides). Finally, the molecular basis of substrate specificity, determined from the proposed research, will mark a breakthrough in the research of acyl-CoA carboxylase. PUBLIC HEALTH RELEVANCE: This project will result in the production of new polyketides that are synthesized with new building blocks. Because polyketides contain many antibiotic and anticancer compounds, the outcome of this project will benefit the general public health by providing new "unnatural" natural products for new drug leads.

描述（由申请人提供）：酰基辅酶A羧化酶（ACCases），例如乙酰辅酶A羧化酶（ACC）和丙酰辅酶A羧化酶（PCC），催化乙酰辅酶A和丙酰辅酶A的羧化以提供丙二酰基和甲基丙二酰基分别为-CoA。这种羧化反应在生物系统中无处不在，因为它使乙酰辅酶A和丙酰辅酶A参与脂肪酸、聚酮化合物和克雷布循环中间体的生物合成。尽管关于 ACCase 的遗传分析、机制和仿生学研究已有完善的知识体系，但由于缺乏关于 ACCase 如何识别其相应底物或抑制剂的分子信息，ACCase 相关疗法的发展受到严重阻碍。我们的长期目标是产生基于 ACCase 的疗法并筛选其药物活性。这一特定应用的目的是确定天蓝色链霉菌 ACC 和 PCC 底物特异性的分子基础，这是我们实现长期目标的下一步。 S. coelicolor ACCases 为聚酮化合物的生物合成提供了延伸单元，聚酮化合物是一类天然产物，包括许多抗生素、抗癌和降胆固醇药物。突变的 ACCas 可能为聚酮生物合成提供新的构建模块，从而可以生物合成具有改变的延伸单元的新聚酮化合物。这些具有抗生素化学模板的新型聚酮化合物将成为针对细菌和病毒的生物恐怖主义目标进行筛选的优秀药物先导物。中心假设是应该可以使用诱变来改变 ACCase 的底物特异性，以产生用于聚酮化合物生物合成的新延伸单元。我们的假设基于以下观察：1) ACCase 亚基对不同的底物和抑制剂具有不同的特异性； 2) 我们关于 ACCase 2 亚基（AccB 和 PccB）结构和功能的初步数据已经确定了负责分子识别的特定残基。如果假设成立，突变的 ACCases 将产生新的取代的丙二酰辅酶A，它可以作为聚酮化合物生物合成的新延伸单元。我们将追求两个具体目标：目标 1. 求解 ACCB 和 PCCB 的共晶结构： 1.1。解析蛋白质-底物共晶结构。 1.2.解决蛋白质调节剂共晶体结构。目标 2. 制作 ACCB 和 PCCB 的活性位点突变体：2.1。系统地突变残基422。2.2。突变酰基辅酶A 结合袋中的残基。 2.3.使生物素结合袋中的残基发生突变。一旦我们确定了可以突变以改变 ACCase 特异性的残基，就有可能生成新的、取代的丙二酰辅酶A，它可以作为聚酮化合物生物合成的新延伸单元。这种创新方法以前从未采用过。由于我们的研究重点和互补的专业知识，我们的研究环境特别有利于成功完成拟议的 ACCases 调查。本申请中提出的研究意义重大，因为其结果使我们能够剖析负责 ACCase 底物特异性的分子特征。从长远来看，该提案的结果将对新抗生素的开发产生显着的积极影响，这些新抗生素要么是 ACCase 抑制剂（用于阻断细菌的脂肪酸生物合成），要么具有新的扩展单元（用于新聚酮化合物的生物合成）。最后，本研究确定的底物特异性的分子基础将标志着酰基辅酶A羧化酶研究的突破。公共卫生相关性：该项目将生产用新结构单元合成的新型聚酮化合物。由于聚酮化合物含有多种抗生素和抗癌化合物，该项目的成果将通过为新药先导物提供新的“非天然”天然产物，造福广大公众健康。