Dissecting the substrate specificity of acyl-CoA carboxylase

剖析酰基辅酶A羧化酶的底物特异性

• 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
• 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

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(申请人提供)：乙酰辅酶A羧基酶(ACCase)，例如乙酰辅酶A羧基酶(ACC)和丙酰辅酶A羧基酶(PCC)，分别催化乙酰辅酶A和丙酰辅酶A的羧化反应，生成丙二酰辅酶A和甲基丙二酰辅酶A。这种羧化反应在生物系统中是普遍重要的，因为它使乙酰辅酶A和丙酰辅酶A参与脂肪酸、聚酮和Kreb环中间体的生物合成。虽然在ACCase的遗传分析、机制和仿生研究方面已经有了成熟的知识体系，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，与ACCase相关的治疗学的发展受到严重阻碍。我们的长期目标是产生基于ACCase的治疗药物，并筛选它们的药物活性。这一特殊应用的目标是确定天蓝色链霉菌ACC和PCC底物专一性的分子基础，这是我们迈向长期目标的下一步。天蓝色链球菌为聚酮的生物合成提供了延伸单元，聚酮是一类天然产品，包括许多抗生素、抗癌和降胆固醇药物。突变的ACCase可能为聚酮的生物合成提供新的构件，从而使具有改变延伸单元的新的聚酮可以被生物合成。这些带有抗生素化学模板的新多酮将是筛选针对细菌和病毒的生物恐怖主义靶标的优秀药物先导。中心假设是，应该可以利用突变来改变ACCase的底物专一性，以产生用于聚酮生物合成的新的延伸单元。我们的假设是基于这样的观察：1)ACCase亚基对不同的底物和抑制剂有不同的特异性；2)我们对ACCase 2-亚基(ACCB和PCCB)的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ACCase将产生新的取代丙二酰辅酶A，它可以作为聚酮生物合成的新延伸单元。我们将追求两个具体目标：目标1。解决ACCB和PCCB的协同结构：1.1。解决蛋白质-底物共晶体结构。1.2.解决蛋白质调节蛋白的共晶体结构。目的2.使ACCB和PCCB：2.1的活性位点发生突变。系统地突变残基422。2.2.酰基-辅酶A结合口袋中的突变残基。2.3.生物素结合口袋中的突变残留物。一旦我们确定了可以突变以改变ACCase特异性的残基，就有可能产生新的、取代的丙二酰COA，作为聚酮生物合成的新的延伸单元。这种创新的方法以前从未被采用过。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助于成功完成拟议的ACCase调查。在本申请中提出的研究具有重要意义，因为它的结果使我们能够剖析导致ACCase底物特异性的分子特征。从长远来看，这一提议的结果将对新抗生素的开发产生重大的积极影响，这些抗生素要么是ACCase抑制剂(用于阻止细菌的脂肪酸生物合成)，要么是具有新的延伸单元(用于新的聚酮的生物合成)。最后，根据所提出的研究确定的底物专一性的分子基础，将标志着酰辅酶A羧基酶研究的突破。 与公共卫生相关：该项目将导致生产新的聚酮，这些聚酮是用新的构建块合成的。由于多酮类化合物中含有许多抗生素和抗癌化合物，该项目的成果将为新的药物先导提供新的“非天然”天然产物，从而造福于公众健康。