Dissecting the substrate specificity of acyl-CoA carboxylase

描述（由申请人提供）：酰基辅酶A羧化酶（ACCases），如乙酰辅酶A羧化酶（ACC）和丙炔辅酶A羧化酶（PCC），分别催化乙酰辅酶A和丙炔辅酶A羧化，生成丙二酰辅酶A和甲基丙二酰辅酶A。这种羧基化反应在生物系统中是普遍重要的，因为它将乙酰辅酶a和丙酰辅酶a用于脂肪酸、聚酮和克雷布循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有一个发达的知识体系，但由于缺乏accase如何识别其相应底物或抑制剂的分子信息，accase相关治疗方法的发展受到严重阻碍。我们的长期目标是产生基于accase的治疗方法并筛选其药物活性。这一特殊应用的目的是确定来自色链霉菌的ACC和PCC的底物特异性的分子基础，这是我们长期目标的下一步。coelicolor accase为多酮类化合物的生物合成提供扩展单元，多酮类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。突变型accase可能为聚酮生物合成提供新的构建模块，因此可以生物合成具有改变延伸单元的新聚酮。这些具有抗生素化学模板的新型聚酮类化合物将成为筛选细菌和病毒生物恐怖目标的优良药物先导物。中心假设是，应该有可能使用诱变来改变ACCase的底物特异性，以产生用于聚酮生物合成的新的延伸剂单位。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。如果这一假设成立，突变的accase将产生新的取代的丙二酰辅酶a，它可以作为聚酮生物合成的新的延伸单元。我们将追求两个具体目标：求解accb和pccb的共晶结构：1.1。解决蛋白质-底物共晶结构。1.2. 解决蛋白质调节共晶结构。目标2。制造accb和pccb的活性位点突变体：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. 突变生物素结合袋中的残基。一旦我们确定了可以突变改变ACCase特异性的残基，就有可能产生新的取代的丙二酰辅酶a，作为聚酮生物合成的新扩展单元。这种创新的做法以前从未采取过。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对ACCases的拟议调查。本应用程序中提出的研究是重要的，因为它的结果使我们能够解剖负责ACCases底物特异性的分子特征。从长远来看，这一建议的结果将对开发新的抗生素产生重大的积极影响，这些抗生素要么是ACCase抑制剂（用于阻断细菌的脂肪酸生物合成），要么是具有新的扩展单元（用于新的聚酮生物合成）。最后，从本研究中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。

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.