CRYSTALLOGRAPHIC STUDIES OF CONFORMATIONAL CHANGES IN ADENYLATE-FORMING ENZYMESE

点击翻译按钮获取中文摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Work in our lab is studying two groups of enzymes, A) adenylate-forming enzymes of a large family that includes acyl-CoA synthetases/ligases and adenylation domains of non-ribosomal peptide synthetases (NRPSs) as well as B) additional NRPS-related enzymes involved in bacterial siderophore synthesis. The adenylate-forming enzymes catalyze a two-step reaction: an initial adenylation reaction to active a carboxylate substrate followed by a second thioester-forming reaction in which the acyl-CoA or acyl-pantetheine thioester is formed. We have determined structures of members of this enzyme family in two conformations that demonstrate a dramatic 140-degree domain rotation between the two half-reactions. We are examining this further to explore enzymes that catalyze only the adenylation half-reaction (AAE from M. thermoautotrophicus) as well as studying members of this family that may serve as antibiotic targets (MbtA). In a collaboration with Dr. Courtney Aldrich, we will determine the structure of MbtA bound to a number of small molecule inhibitors that he has designed and synthesized. We have native data to 2.0A for AAE and will collect data on SeMet labeled protein crystals at CHESS. We have crystallized MbtA however these are in early stages of characterization. The second field of our work concerns other enzymes involved in other siderophore (iron-sequestering) compounds synthesized by NRPSs and related enzymes. We have determined structures of two proteins involved in the synthesis of pyoverdine, a siderophore from the Cystic Fibrosis-related pathogen, P. aeruginosa. One was solved using data from CHESS. These structures are part of a larger structure/function to identify the synthetic pathway for pyoverdine and to identify potential targets for anti-infective agents that could block pyoverdine production. Similarly, we have marginal native data (2.6A) on one of these pyoverdine synthetic enzyme, PvcA, and plan to collect improved native, as well as Se MAD data on the requested trip. Several other samples, for which we have determined native structures, will be studied to obtain additional liganded structures, as described in the Specimen Section (#5).