RUI: Regulation of Diverse Bacterial ADPGlucose Pyrophosphorylases

Meyer这项研究的重点是动力学，物理，分子和代谢研究的独特调节细菌ADPG Ppases，在糖原和淀粉生物合成途径的限速酶，从以下来源：Rhodobacter sphaeroides，Rhododocellum rubrum，根癌农杆菌，和Rhodobacter capsulatus。ADPG Ppase的活性受各种变构效应分子的结合调节，这取决于生物体的碳利用途径。 对这个酶家族进行完整的分子比较将使我们能够以增强功能为目标进行合理的蛋白质工程。 ADPG Ppase的成功工程改造将允许在转基因植物中过量生产淀粉。 本研究项目的具体目标是：I：鉴定对这些ADPG Ppases的功能、调节和物理特性重要的氨基酸; II：独特细菌ADPG Ppases基因的克隆和测序; III：ADPG Ppases的表达和蛋白质工程;以及，长期而言，IV：阐明变体ADPG Ppases和糖原/淀粉代谢酶对终产物淀粉结构的影响。 这种跨学科的方法将允许出现结构/功能关系的详细图片。 合理诱变的信息（目的III.）将部分来自目标一和目标二的结果。 用于鉴定和表征各种活性和变构激活剂和抑制剂位点中的氨基酸的技术包括酶动力学、化学修饰、有限蛋白水解、X射线晶体学、基因的克隆和测序（包括使用PCR）、蛋白质表达、生物信息学（核酸和蛋白质序列的比对）以及定点和随机诱变。 长期目标包括突变体和天然晶体结构之间的比较，同时对突变体进行动力学和其他功能分析。 开发中的实验还将涉及在体内和体外重组系统中利用工程化的ADPG酶与各种淀粉酶、分支酶和脱支酶组合，以阐明终产物淀粉的结构/功能关系。 从这些系统中产生的葡聚糖将被分离并分析产量、链长和支化模式。由于各种行业对天然和改性淀粉的需求不断增加，糖原和淀粉生物合成途径的调节越来越受到关注。 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材料，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，该项目非常适合在生物化学，分子生物学和生物技术的理论和实践方面培养学生。 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.