紫色非硫光合菌之磷酸化蛋白質體學研究

紫色非硫光合菌Rhodopseudomonas palustris (R.palustris) 廣泛分布於土壤和河水中，分類階層屬於alpha-proteobacteria。R. palustris能利用太陽光當作能量來源，也可吸收大氣中的二氧化碳轉變成本身的重量，除此之外，它還能代謝並回收植物的木質組織。由於R. palustris和碳的利用與回收有密切的關係，因此被DOE Carbon Management Program選為基因體定序的物種。R. palustris能夠生長在光合異營、光合自營、化學異營以及化學自營等不同的代謝環境，並且可能利用磷酸化的方式來調控不同代謝路徑的轉變。在本研究中，我們在化學異營以及光合異營的條件下，利用shotgun的方式分析R. palustris的磷酸化蛋白質體表現。我們在化學異營的條件下鑑定到80條磷酸化胜肽，其可對應到73個蛋白質；而在光合異營的條件下則有74條磷酸化胜肽，可對應到68個蛋白質。另外，針對磷酸化胜肽定量的結果中發現，pyruvate phosphate dikinase (PPDK, RPA1051) 當中的胜肽“GGMpTSHAAVVAR”，在光合異營下蘇胺酸的磷酸化程度比化學異營條件下高出兩倍以上。目前已知PPDK在C4及CAM植物中是受磷酸化調控活性，並且在固碳上扮演重要的角色，但磷酸化的PPDK在原核生物的研究當中目前仍是未知。在我們的光合異營及化學異營內生性PPDK酵素活性測試實驗中發現，PPDK在光合異營條件下的活性較高；另外，在我們點突變磷酸化位置的實驗中也顯示，蘇胺酸的磷酸化位置對PPDK的活性是重要的。因此，從我們的結果推測，PPDK受到光刺激後，使蘇胺酸磷酸化並且同時具有活性，而磷酸化的PPDK也可能調控了R. palustris在光合異營與化學異營間的轉換機制。

關鍵字

紫色非硫光合菌；磷酸化蛋白質體學；磷酸化胜肽光合異營；化學異營

並列摘要

Rhodopseudomonas palustris (R. palustris) is a purple nonsulfur anoxygenic phototrophic bacterium that belongs to the alpha-proteobacteria. It is a common soil and water bacterium that makes its living by converting sunlight to cellular energy and by absorbing atmospheric carbon dioxide and converting to biomass. This microbe can also degrade and recycle components of the woody tissues of plants. Because of its intimate involvement in carbon management and recycling, R. palustris was selected by the DOE Carbon Management Program for genome sequencing. R. palustris exhibits the ability to grow under photoheterotrophic, photoautotrophic, chemoheterotrophic and chemoautotrophic conditions, and can switch between the four different modes of metabolism for survival by protein phosphorylation. Here, we analyzed the phosphoproteome of R. palustris in photoheterotrophic condition with a shotgun approach and identified 80 phosphopeptides from 73 phosphoproteins and 74 phosphopeptides from 68 phosphoproteins at chemohetrotrophic and photoheterotrophic condition, respectively. Our results revealed that the threonine phosphorylated peptide “GGMpTSHAAVVAR” from pyruvate phosphate dikinase (PPDK, RPA1051) in photoheterotrophic condition is elevated more than 2 folds than in chemoheterotrophic condition. PPDK performs a light-dependent activity and plays an important role in carbon fixation in C4 and CAM plants. However, the function of phosphorylated PPDK is still unknown in prokaryotic cell. Here, we showed that PPDK enzyme activity is higher in photoheterotrophic than in chemoheterotrophic. In our in vitro point mutation experiment revealed that threonine phosphorylation site played an important role in regulating PPDK activity. We suggested that light could stimulate threonine phosphorylation of PPDK and might enhance its activity which could regulate the switch mechanism of R. palustris in photoheterotrophic and chemoheterotrophic conditions.

並列關鍵字

Rhodopseudomonas palustris ； phosphoproteome ； phosphopeptide ； photoheterotrophic ； chemoheterotrophic

參考文獻

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紫色非硫光合菌之磷酸化蛋白質體學研究

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