Characterization of novel sulfur salvage mechanisms in Rodospirillum rubrum

描述(由申请人提供)：回收末端的、含硫的代谢副产物是几乎所有生物体的基本过程。细胞在多胺合成过程中使用S-腺苷蛋氨酸(SAM)进行细胞信号传递、生长和增殖，从而导致最终的有毒副产物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代谢回蛋氨酸以实现细胞正常功能的挑战。许多癌症表现出MTA代谢受损，导致MTA积聚，从而刺激或抑制癌症的进展。最近，MTA磷酸化酶已成为癌症治疗的靶点，对MTA水平的调节被发现可以控制肿瘤的增殖。然而，靶向MTA代谢途径下游的基因对蛋氨酸和SAM挽救的影响却知之甚少。因此，必须确定代谢MTA以支持适当的细胞生长和信号传递的机制。在细菌中，对肺炎克雷伯氏菌和枯草芽孢杆菌中的硫回收进行了适度的描述。然而，这两种生物似乎并不包含已进化出的众多且可能更为普遍的硫磺回收机制。红色罗氏螺杆菌是阐明和表征蛋氨酸回收途径的理想系统。在好氧条件下，Rubrum使用一种类似Rubisco的蛋白质来循环MTA，而在厌氧条件下，Rubisco以不同的和独立的途径使用。这既是首次观察到的厌氧抢救案例，也是Rubisco在硫代谢中的应用。在这项工作中，我们将使用基因敲除菌株分析和转录组图谱相结合的方法来识别直接参与Rubisco介导的MTA代谢途径的结构基因。同时，我们将使用重组酶分析以及高效液相色谱和核磁共振波谱的代谢物分析来确定在Rubisco介导的途径中观察到的每个基因产物的酶底物和产物。由此，我们将能够充分描述以前未知的厌氧代谢途径，即红色葡萄球菌利用Rubisco循环MTA的途径。这将提供对Rubisco如何参与MTA代谢以支持适当的SAM依赖的细胞信号、生长和增殖的机械性理解。此外，这将提供对显示MTA代谢和蛋氨酸挽救途径受损的癌症病理的洞察。

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.