RUI: Regulation of Diverse Bacterial ADPGlucose Pyrophosphorylases

Meyer本研究集中于对唯一调节的细菌ADPG Ppase的动力学、物理、分子和代谢的研究，ADPG Ppase是糖原和淀粉生物合成途径中的限速酶，来自以下来源：球形红杆菌、红色红螺菌、根癌农杆菌和胶囊红杆菌。ADPG Ppase的活性受各种变构效应分子结合的调节，依赖于生物体的碳利用途径。对这一酶家族进行完整的分子比较，将使我们能够进行合理的蛋白质工程，以增强功能。ADPG Ppase工程的成功将允许在转基因植物中过量生产淀粉。这一研究项目的具体目标是：鉴定对这些ADPG Ppase的功能和调节以及物理特性至关重要的氨基酸；II：克隆和测序细菌特有的ADPG Ppase基因；III：ADPG Ppase的表达和蛋白质工程；以及IV：阐明变异的ADPG Ppase和糖原/淀粉代谢酶对最终产品淀粉结构的影响。这种跨学科的方法将允许出现结构/职能关系的详细图景。合理诱变的信息(Aim III.)用于鉴定和表征各种活性和变构激活剂和抑制物部位(S)的氨基酸的技术包括酶动力学、化学修饰、有限的蛋白质分解、X射线结晶学、基因克隆和测序(包括聚合酶链式反应)、蛋白质表达、生物信息学(核酸和蛋白质序列的比对)以及定点和随机突变。长期目标包括对突变体和天然晶体结构进行比较，同时对突变体进行动力学和其他功能分析。正在开发的实验还将涉及利用体内和体外重组系统中的工程ADPG Ppase，结合各种淀粉合成酶、分支和去分支酶来阐明最终产品淀粉的结构/功能关系。从这些系统中产生的葡聚糖将被分离出来，并对产量、链长和分支模式进行分析。由于各种行业对天然淀粉和变性淀粉的需求不断增加，糖原和淀粉生物合成途径的调节是一个日益重要的领域。这些可再生和可生物降解的碳源可以作为生物乙醇、有机酸和抗生素合成的廉价起始原料，并在制造特种塑料、粘合剂、洗涤剂、表面活性剂和包装材料方面具有巨大的潜力。除了对酶学和农业生物技术做出贡献外，该项目还非常适合于在以本科为主的机构培训学生生物化学、分子生物学和生物技术的理论和实践。学生在实验室获得的背景和经验使他们成为学术和工业职位的有吸引力的候选人。

Meyer This research is focused on kinetic, physical, molecular, and metabolic studies of the uniquely regulated bacterial ADPG Ppases, the rate-limiting enzymes in the glycogen and starch biosynthetic pathways, from the following sources: Rhodobacter sphaeroides, Rhodospirillum rubrum, Agrobacterium tumefaciens, and Rhodobacter capsulatus. The activity of ADPG Ppase is modulated by the binding of various allosteric effector molecules depending on the carbon utilization pathway of the organism. A complete molecular comparison of this family of enzymes will allow us to perform rational protein engineering with the goal of enhancing function. The successful engineering of ADPG Ppase would allow for the overproduction of starch in transgenic plants. The specific aims of this research project are I: Identification of the amino acids important for function and regulation and physical characterization of these ADPG Ppases; II: Cloning and sequencing of unique bacterial ADPG Ppase genes; III: Expression and protein engineering of ADPG Ppases; and, longer term, IV: Elucidating the effect of variant ADPG Ppases and glycogen/starch metabolism enzymes on the structure of the end-product starch. This interdisciplinary approach will allow for a detailed picture of structure/function relationships to emerge. Information for rational mutagenesis (Aim III.) will be derived in part from the results of Aims I and II. The techniques utilized in order to identify and characterize the amino acids in the various active and allosteric activator and inhibitor site(s) include enzyme kinetics, chemical modification, limited proteolysis, X-ray crystallography, cloning and sequencing of genes (including use of PCR), protein expression, bioinformatics (alignment of nucleic acid and protein sequences), and site-directed and random mutagenesis. Long-range goals include a comparison between mutant and native crystal structures in parallel with kinetic and other functional analyses of the mutants. Experiments in development will also involve utilizing engineered ADPG Ppases in both in vivo and in vitro recombinant systems in combination with various starch synthases, branching, and debranching enzymes to elucidate structure/function relationships of the end product starch. The glucan produced from these systems will be isolated and analyzed with respect to yield, chain length, and branching pattern.The regulation of the glycogen and starch biosynthetic pathways is a growing area of interest due to the increasing demand for natural and modified starches in a variety of industries. These renewable and biodegradable carbon sources can serve as inexpensive starting materials for bio-ethanol, organic acids, and antibiotic synthesis and have great potential for use in the making of specialty plastics, adhesives, detergents, surfactants, and packaging materials. Beyond contributing to enzymology and agricultural biotechnology, this project is well suited to training students at a primarily undergraduate institution in the theory and practice of biochemistry, molecular biology, and biotechnology. The background and experience students gain in the laboratory makes them attractive candidates for both academic and industrial positions.