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（由申请人提供）：酰基辅酶A羧酸酶（ACCASE），例如乙酰辅酶A羧化酶（ACC）和丙酰辅酶A羧化酶（PCC），催化乙酰基羧基化和丙酰辅酶A的羧化，以分别提供malonyl--和甲基甲基甲酰基-COA。这种羧化反应在生物系统中无处不在，因为它将乙酰辅酶A和丙酰辅酶A定为脂肪酸，Polyketides和Kreb循环中间体的生物合成。尽管在遗传分析，机理和仿生研究方面有良好的知识，但由于缺乏有关accas识别其相应底物或抑制剂的分子信息，因此严重阻碍了与Accase相关疗法的发展。我们的长期目标是生成基于ASCASE的治疗剂并筛选其药物活动。这种特定应用的目的是朝着我们的长期目标迈出的下一步，是确定来自Coelicolor链霉菌和PCC的底物特异性的分子基础。 Coelicolor Scass为聚酮化合物的生物合成提供了扩展单位，PoloteTiess是一类天然产物，其中包括许多抗生素，抗癌和降低胆固醇的药物。突变型式可潜在地为聚酮化合物的生物合成提供新的构建块，以便可以将具有变化的扩展器单元的新聚酮化合物生成生物合成。这些新的聚酮化合物具有抗生素化学模板，将是极好的药物导致对细菌和病毒的生物恐怖靶标的筛查。 中心假设是，应该使用诱变来改变ASCASE的底物特异性，以生成用于聚酮化合物生物合成的新扩展单元。我们基于以下观察结果，即1）Accase亚基对不同的底物和抑制剂具有不同的特异性； 2）我们有关Accase 2-subunits（ACCB和PCCB）的结构和功能的初步数据已经鉴定出负责分子识别的特定残基。如果该假设是正确的，则突变体的空间将产生新的取代的malonyl-coas，可以用作聚酮化合物生物合成的新扩展单元。我们将追求两个具体的目标：目标1。求解ACCB和PCCB的共晶结构：1.1。求解蛋白质 - 基层共晶结构。 1.2。解决蛋白质调节剂共晶结构。 AIM 2。使ACCB和PCCB的主动位点突变体：2.1。系统突变的残基422。2.2。酰基辅酶A结合袋中的突变残基。 2.3。生物素结合口袋中的突变残基。 一旦我们确定了可以突变以改变ACACASE的特异性的残基，就有可能产生新的，取代的丙二酰果-COA，这些丙二酰果会可以用作Polyketide Biosynsiss的新扩展单元。这种创新的方法以前尚未采用。由于我们的研究重点和互补的专业知识，我们的研究环境对于成功完成拟议的Accases调查而尤其具有导电性。在本应用中提出的研究很重要，因为它的结果使我们能够剖析负责Accass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发展产生重大积极影响，这些抗生素要么是ACCASE抑制剂（用于阻断脂肪酸的生物合成）或具有新的扩展单元（用于新的Polyketides的生物合成）。最后，根据拟议的研究确定的底物特异性的分子基础将标志着酰基辅酶A羧化酶的研究的突破。 公共卫生相关性：该项目将导致生产与新的构件合成的新聚酮化合物。由于聚酮化合物含有许多抗生素和抗癌化合物，因此该项目的结果将通过为新药铅提供新的“不自然”天然产品来受益。