Functional Genomics of D-Amino Acid Metabolism in Pseudomonas Aeruginosa

智力美德D-氨基酸在所有生物体中自然合成，以发挥各种特定功能。例子包括众所周知的D-Ala和D-Glu作为细菌细胞壁的基本成分，到最新发现的D-Ser作为人类的神经递质。细胞内D-氨基酸的水平受到严格的调节，以防止任何潜在的有害影响。迄今为止，与L-氨基酸相比，D-氨基酸催化剂的生物化学尚未得到深入研究，因此，信息仍然是零碎的。铜绿假单胞菌以其巨大的分解代谢能力而闻名，能够利用许多D-氨基酸作为营养物质，因此可以作为探索D-氨基酸代谢新途径和新酶的极好模式生物。最好的证据是最近的一份报告，PI的小组在以前的NSF资助的项目中发现了一种新型的D-到-L精氨酸外消旋化，这种外消旋化是通过偶联分解代谢和合成代谢酶实现的。初步研究的结果还揭示了D-氨基酸利用的几个重要特征。D-氨基酸具有两种潜在的生理功能-转化为L-氨基酸用于肽合成或降解以用作氮源或碳源。如何利用这些D-氨基酸中的每一种取决于特定消旋酶或分解代谢酶/途径的存在以及诱导水平。本研究的目的是继续努力探索D-氨基酸的代谢途径与系统的方法。本项目的具体目标是：（i）阐明DadAX和DauAB在D-氨基酸脱氨基和外消旋中的生理功能和生化特性;（ii）探索D-谷氨酸/谷氨酰胺/天冬酰胺和D-脯氨酸/羟脯氨酸的分解代谢途径。将进行实验以测试由此鉴定的基因及其编码酶的拟议生理和生化功能。同时，将进行更系统的转录组分析，以更好地了解细胞对这些D-氨基酸的反应。因此，从这项研究中确定的基因和编码酶将进行进一步的表征，相应的生理功能，遗传调控，和生化特性的D-氨基酸催化剂。 到目前为止，只有有限的努力已致力于系统地探讨生化多样性的D-氨基酸。这项研究的结果预计将进一步扩展目前的氨基酸代谢知识。更广泛的影响PI计划继续努力，保持一个积极的研究环境，由博士后助理，MS和博士研究生，和本科生。此外，PI将通过在夏季担任McNair计划的教师导师来扩大招募有前途的少数民族学生进行研究的努力。作为科学家，PI实验室将提供独特的培训机会，结合现代基因组学/蛋白质组学和细菌生理学和生物化学领域的传统遗传学/生物化学方法。此外，PI与蛋白质晶体学和机械酶学同事之间的持续合作将通过定期联合小组会议和鼓励学生参加合作同事提供的课程来促进不同学科的学员受益。这项拟议研究的成功将在培养具有多学科覆盖面的竞争力的研究人员和招募代表性不足的少数民族学生进入生物研究职业方面产生更广泛的影响。卢实验室的所有成员将参加地方，国家和国际研究研讨会，以传播实验室的研究和教育活动，以更广泛的科学界。

Intellectual MeritD-amino acids are naturally synthesized in all living organisms to serve in a variety of specific functions. Examples include the well-known D-Ala and D-Glu as essential components of bacterial cell wall to the latest discovery of D-Ser as neurotransmitter in humans. The level of D-amino acids inside the cells is subjected to tight regulation to prevent any potential harmful effects. Up to date, the biochemistry of D-amino acid catabolism has not been intensively studied in comparison to those of L-amino acids, and therefore, the information is still fragmentary. Famous for its enormous catabolic capacity, P. aeruginosa is able to utilize many D-amino acids as nutrient, and hence serves as an excellent model organism to explore novel pathways and enzymes for D-amino acid metabolism. It is best evidenced by a recent report of a new type of D-to-L arginine racemization by coupled catabolic and anabolic dehydrogenases discovered by the PI's group on a previous NSF-funded project. The results of preliminary studies also uncovered several important features in D-amino acid utilization. D-amino acids have two potential physiological functions - inversion to L-amino acids for peptide synthesis or degradation to serve as nitrogen or carbon source. How each of these D-amino acids can be utilized depends on the presence as well as the induction level of specific racemases or catabolic enzymes/pathways. The goal of this research is to continue efforts in exploration of metabolic pathways for D-amino acids with a systemic approach. The specific aims of this project are: (i) to elucidate physiological functions and biochemical properties of DadAX and DauAB in D-amino acid deamination and racemization; and (ii) to explore catabolic pathways for D-glutamate/glutamine/asparagine and D-proline/hydroxyproline. Experiments will be conducted to test the proposed physiological and biochemical functions of thus identified genes and their encoding enzymes. Meanwhile, a more systemic transcriptome analysis will be conducted to have a better understanding of cellular responses to the presence of these D-amino acids. Genes and encoding enzymes thus identified from this study will be subjected to further characterization accordingly regarding physiological functions, genetic regulation, and biochemical properties in D-amino acid catabolism. Up till now, only limited effort has been devoted systemically to explore the biochemical diversity toward D-amino acids. Findings from this research are anticipated to further extend the current knowledge of amino acid metabolism.Broader ImpactsThe PI plans to continue the effort in maintaining an active research environment composed of post-doc associates, MS and PhD graduate students, and undergraduate students. In addition, the PI will extend the effort for recruiting promising minority students to research by serving as faculty mentor for the McNair Program in the summers. Scientifically, the PI lab will provide unique training opportunities with a combination of modern genomics/proteomics and conventional genetics/biochemistry approaches in the area of bacterial physiology and biochemistry. In addition, the ongoing collaborations between PI and colleagues in protein crystallography and mechanistic enzymology will be fostered to benefit trainees of different disciplines, by way of regular joint group meetings and encouragement of students taking courses offered by collaborating colleagues. The success of this proposed research will have broader impacts in training competitive researchers with multi-disciplinary coverage and in recruiting under-representative minority students into careers of biological research. All members of the Lu laboratory will participate in local, national, and international research symposia to disseminate the research and educational activities of the laboratory to a broader scientific community.