Understanding the physiological context of the metabolic needs of a pathogen provides a framework for targeted development of antimicrobials. In the case of Salmonella enterica, respiration of ethanolamine in the intestinal environment provides a fitness advantage over commensal microorganisms. Ethanolamine catabolism is a coenzyme B12-dependent pathway that occurs inside a proteinaceous compartment called the ethanolamine (Eut) metabolosome. Ethanolamine is deaminated by ethanolamine ammonia lyase (EAL) to form acetaldehyde, which eventually enters central metabolism as acetyl-CoA. EutA reactivates inactive EAL by removing dysfunctional coenzyme B12 at the expense of ATP. These reactions are needed to trigger the initial step of ethanolamine catabolism hence understanding the interactions required for these processes in the context of the Eut metabolosome would provide opportunities for targeted disruption of the metabolic pathway. There are several gaps of knowledge that need to be filled in so we can improve our understanding f ethanolamine catabolism in this human pathogen. First, the mechanism of catalysis of the adenosyltransferase EutT enzyme that converts vitamin B12 to coenzyme B12 is unknown, thus will be investigated. While the S. enterica EutT is a metalloprotein, EutT homologues in some other pathogens (e.g., Listeria monocytogenes, Clostridum tetani) function without a metal. Metal containing and metal-less Eut enzyme will be studied, and their mechanisms of catalysis compared to elucidate the role of the metal center. Second, it is not understood how coenzyme B12 is delivered to EAL from EutT. Preliminary evidence strongly suggests that EutA mediates the delivery, and that EutT, EutA and EAL may form a complex. A multidisciplinary approach (crystallography, spectroscopy, molecular biology, biochemistry, in vivo genetics, and physiology) will be used to address this complex problem. Collectively, this work will advance our understanding of how cells synthesize and deliver essential coenzymes to the enzymes that use them, in this case inside a cellular compartment.
. Coenzyme B12 is only synthesized by prokaryotes, including many pathogens, and it is essential to the survival of animals, including humans. The synthesis and delivery of coenzyme B12 to the enzyme that uses it to breakdown ethanolamine is poorly understood. The project will address these questions using a combination of biochemical, biophysical, genetic and physiological approaches. The proposed work will provide insights into the physiology of several human pathogens.
| Costa, Flavia G; Escalante-Semerena, Jorge C (2018) A New Class of EutT ATP:Co(I)rrinoid Adenosyltransferases Found in Listeria monocytogenes and Other Firmicutes Does Not Require a Metal Ion for Activity. Biochemistry 57:5076-5087 |
| Stracey, Nuru G; Costa, Flavia G; Escalante-Semerena, Jorge C et al. (2018) Spectroscopic Study of the EutT Adenosyltransferase from Listeria monocytogenes: Evidence for the Formation of a Four-Coordinate Cob(II)alamin Intermediate. Biochemistry 57:5088-5095 |
