Collaborative Research: Expanding the repertoire of chemical tools to study and characterize bacterial Gcn5-related N-acetyltransferase functions

这项国家科学基金本科院校研究奖由生命过程化学计划资助旧金山州立大学的Misty L.Kuhn博士和芝加哥Loyola大学的Daniel P.Becker博士创建生化工具，以研究土壤微生物铜绿假单胞菌中功能未知的Gcn5相关N-乙酰转移酶(GNAT)蛋白家族中的酶。具体地说，这些工具与通过X射线结晶学进行的结构测定结合使用，以更好地了解分子如何与GNAT结合。由于对铜绿假单胞菌的蚊虫结构/功能关系知之甚少，这项工作填补了这一知识空白。确定这些功能很重要，因为人类福利依赖于健康的农业。这个项目通过建立第一个蛋白质结晶资源和增加少数族裔和弱势本科生的复杂生物化学技能库来加强SFSU的研究基础设施。本项目的目标是利用蛋白质X射线结晶学、分子建模、有机合成和酶学的组合来改进功能未知的铜绿假单胞菌GATS的功能注释。用生化工具和X射线结晶学研究了几种GNAT蛋白的受体部位的结构。对催化和底物专一性至关重要的活性位点残基通过分子建模、定点突变和酶动力学进行鉴定和测试。该项目的结果为分类实验中未描述的GNAT家族成员和改进未知功能的GNAT的计算功能注释提供了基础知识。

This National Science Foundation Research in Undergraduate Institutions award from the Chemistry of Life Processes Program funds Dr. Misty L. Kuhn from San Francisco State University (SFSU) and Dr. Daniel P. Becker from Loyola University Chicago to create biochemical tools to study enzymes in the family of Gcn5-related N-acetyltransferase (GNAT) proteins of unknown function from the soil microorganism, Pseudomonas aeruginosa. Specifically, these tools are used in combination with structure determinations via X-ray crystallography to gain a greater understanding of how molecules bind to GNATs. Since relatively little is known about the structure/function relationship of GNATs from P. aeruginosa, this work fills this gap in knowledge. Determining these functions is important because human welfare depends on healthy agriculture. This project enhances the research infrastructure at SFSU by establishing its first protein crystallization resource and increasing the repertoire of sophisticated biochemistry skills of underrepresented minority and disadvantaged undergraduate students. The goal of this project is to improve the functional annotation of P. aeruginosa GNATs of unknown function using a combination of protein X-ray crystallography, molecular modeling, organic synthesis, and enzymology. The architecture of the acceptor site of several GNAT proteins are probed using biochemical tools and X-ray crystallography. Active site residues that are critical for catalysis and substrate specificity are identified and tested using molecular modeling, site-directed mutagenesis, and enzyme kinetics. The results of this project provides the foundational knowledge to categorize experimentally uncharacterized members of the GNAT family and improve computational functional annotation of GNATs of unknown function.