Characterization of novel sulfur salvage mechanisms in Rodospirillum rubrum

描述（由申请人提供）：回收死端含硫代谢副产物是几乎所有生物体的基本过程。细胞在多胺合成期间利用S-腺苷甲硫氨酸（SAM）用于细胞信号传导、生长和增殖，这导致死端和毒性副产物5-甲硫基腺苷（MTA）。鉴于有机硫在环境中通常是有限的，细胞面临着将MTA代谢回甲硫氨酸以实现适当细胞功能的挑战。许多癌症表现出受损的MTA代谢，导致MTA的积累，这可以刺激或抑制癌症进展。最近，MTA磷酸化酶已成为癌症治疗疗法的靶标，并且已发现调节MTA水平可控制癌症增殖。然而，关于MTA代谢途径下游的靶向基因对甲硫氨酸和SAM补救的影响知之甚少。因此，必须确定MTA代谢以支持适当细胞生长和信号传导的机制。在细菌中，在肺炎克雷伯氏菌和枯草芽孢杆菌中适度描述了硫补救。然而，这两种微生物似乎并不包括已经进化的众多且可能更普遍的硫补救机制。模式细菌，Rodoacillum rubrum，是一个理想的系统，其中新的蛋氨酸补救途径可以阐明和表征。在有氧条件下，R. rubrum利用RuBisCO样蛋白来回收MTA，而在厌氧条件下，RuBisCO用于不同的单独途径。这既是第一个观察到的厌氧补救案例，也是RuBisCO在硫代谢中的应用。在这项工作中，我们将采用敲除菌株分析和转录组分析相结合，以确定直接参与RuBisCO介导的MTA代谢途径的结构基因。同时，我们将采用重组酶测定结合通过高效液相色谱和核磁共振光谱法进行的代谢物分析，以鉴定在RuBisCO介导的途径中观察到的每种基因产物的酶底物和产物。由此，我们将能够充分表征以前未知的厌氧代谢途径，通过该途径，R。rubrum使用RuBisCO对MTA进行纯化。这将提供RuBisCO如何参与MTA代谢以支持适当的SAM依赖性细胞信号传导、生长和增殖的机制理解。此外，这将提供对表现出受损的MTA代谢和甲硫氨酸补救途径的癌症病理学的深入了解。

DESCRIPTION (provided by applicant): 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.