Salvage of dead-end, sulfur-containing metabolic byproducts is an essential process in nearly all organisms. Cells employ S-adenosylmethionine (SAM) during polyamine synthesis for cell signaling, growth, and proliferation, which results in the dead-end and toxic byproduct, 5-methylthioadenosine (MTA). Given that organic sulfur is typically limiting in the environment, cells are faced with the challenge of metabolizing MTA back into methionine for proper cellular function. Numerous carcinomas exhibit impaired MTA metabolism, resulting in an accumulation of MTA, which can stimulate or repress carcinoma progression. Recently, MTA phosphorylase has been a target for cancer treatment therapies, and regulation of MTA levels has been found to control cancer proliferation. However, little is known about the effects of targeting genes downstream in the MTA metabolism pathway for methionine and SAM salvage. Therefore, the mechanisms by which MTA is metabolized to support proper cellular growth and signaling must be determined. In bacteria, sulfur salvage has been moderately described in Klebsiella pneumoniae and Bacillus subtilis. However, these two organisms do not appear to encompass the numerous and potentially more prevalent sulfur salvage mechanisms that have evolved. The model bacterium, Rodospirillum rubrum, is an ideal system in which novel methionine salvage pathways can be elucidated and characterized. Under aerobic conditions, R. rubrum employs a RuBisCO-like protein to recycle MTA, while under anaerobic conditions RuBisCO is used in a distinct and separate pathway. This is both the first observed case of anaerobic salvage and moreover the use of RuBisCO in sulfur metabolism. In this work, we will employ a combination of knockout strain analysis and transcriptome profiling to identify structural genes directly involved in the RuBisCO-mediated MTA metabolism pathway. Coordinately, we will employ recombinant enzyme assays coupled with metabolite analysis by high- performance liquid chromatography and nuclear magnetic resonance spectroscopy to identify the enzyme substrate and product for each gene product observed in the RuBisCO-mediated pathway. From this we will be able to fully characterize the previously unknown anaerobic metabolic pathway by which R. rubrum recycles MTA using RuBisCO. This will provide mechanistic understanding of how RuBisCO participates in MTA metabolism to support proper SAM-dependent cell signaling, growth, and proliferation. Additionally, this will provide insight into cancer pathologies that exhibit impaired MTA metabolism and methionine salvage pathways.
The human body requires methionine based compounds for proper growth and cellular division. Numerous cancers including breast, lung, colon, kidney, bladder, melanoma, and glioblastoma are defective in methionine metabolism, leading to abnormal cell proliferation. The current study provides novel insights into methionine salvage by fully characterizing heretofore undescribed alternative mechanisms by which cells can recycle methionine for proper growth and metabolism.
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