探討麩氨基硫亞精胺合成酶/水解酶之反應機制與其在 大腸桿菌的氧化還原中所扮演的角色

E. coli Gsp Synthetase/Amidase: Reaction Mechanisms and Its Role in Redox Regulation

江秉諭(Bing-Yu Chiang)

指導教授 ： 林俊宏

國立臺灣大學/生命科學院/生化科學研究所/博士(2010年)

https://doi.org/10.6342/NTU.2010.01871

摘要

有些格蘭氏陰性菌中， 麩氨基硫亞精胺 (glutathionylspermidine, Gsp) 的生成與分解是由一個雙功能性酵素麩氨基硫亞精胺合成酶/水解酶 (glutathionylspermidine synthetase/amidase, GspSA) 所催化。然而，Gsp 在菌體中的生理功能至今仍是不清楚。此外，GspSA 中兩個具有相反活性的區塊如何相互調控目前也是個未知謎團。在本論文中，我們首先提出Gsp 會與大腸桿菌中蛋白質上的某些硫基 (thiol) 形成雙硫鍵。此種新的蛋白質後修飾會隨著外界的氧化壓力的上升而增加。隨後的研究發現，過氧化氫會選擇性的抑制 Gsp水解酶的活性，而不會影響Gsp 合成酶的活性。這選擇性抑制的現象會導致細胞內 Gsp 的累積，並進而造成蛋白質 Gsp thiolation 的上升。利用X 光繞射分析與化學修飾，我們進一步解釋的這個 Gsp amidase 的選擇性抑制是來自於催化胺基酸Cys 59 被氧化成 sulfenic acid。由高解析度X光繞射分析指出GspSA 中有數個特殊的氫鍵來穩定此 sulfenic acid。本研究中提出了一套機制來描述在大腸桿菌中Gsp的如何進行調控，並說明其在氧化還原中所扮演的角色。¬而 Grx-/GspSA- 雙基因突變株顯示出對氧化壓力高度敏感，也說明了 Gsp 在對大腸桿菌在氧化壓力扮演的角色。最後，為了瞭解到底有哪些蛋白質會被修飾，我們藉由被生物素標定的亞精胺與大腸桿菌進行培養。大腸桿菌內的 Gsp 合成酶會合成具有生物素標定的 Gsp 並進一步結合到蛋白質上，藉此親合性純化與質譜分析而得到被 Gsp 標定蛋白質的身分與位置。

關鍵字

麩氨基硫亞精胺

並列摘要

Certain bacteria synthesize glutathionylspermidine (Gsp), from glutathione (GSH) and spermidine. E. coli Gsp synthetase/amidase (GspSA) catalyzes both the synthesis and hydrolysis of Gsp. Prior to the work reported herein, the physiological role(s) of Gsp or how the two opposing GspSA activities are regulated had not been elucidated. We report that Gsp-modified proteins from E. coli contain mixed disulfides of Gsp and protein thiols, standing for a new type of post-translational modification formerly undocumented. The level of these proteins is increased by oxidative stress. We attribute the accumulation of such proteins to the selective inactivation of GspSA amidase activity. X-ray crystallography and a chemical modification study indicated that the catalytic cysteine thiol of the GspSA amidase domain is transiently inactivated by H2O2 oxidation to sulfenic acid, which is stabilized by a very short hydrogen bond with a water molecule. We propose a set of reactions that explains how the levels of Gsp and Gsp S-thiolated proteins are modulated in response to oxidative stress. The hypersensitivities of GspSA/glutaredoxin null mutants to H2O2 support the idea that GspSA and glutaredoxin act synergistically to regulate the redox environment of E. coli. Additionally, a platform based on metabolic incorporation of biotinated spermidine was developed to identify Gsp Sthiolated proteins.

並列關鍵字

glutathionylspermidine

參考文獻

1. Fang, F. C. (2004) Nat Rev Microbiol 2, 820-832

2. Masip, L., Veeravalli, K., and Georgiou, G. (2006) Antioxid Redox Signal 8,753-762

3. Meister, A., and Anderson, M. E. (1983) Annu Rev Biochem 52, 711-760

11. Ritz, D., and Beckwith, J. (2001) Annu Rev Microbiol 55, 21-48

15. Krauth-Siegel, R. L., and Ludemann, H. (1996) Mol Biochem Parasitol 80, 203-208