探討比菲德氏龍根菌LNB/GNB代謝基因組中磷酸解酶及激酶之蛋白複合結構與催化機制

Structure-based catalytic mechanisms of galacto-N-biose/lacto-N-biose I phosphorylase and N-acetylhexosamine 1-kinase in the LNB/GNB pathway of Bifidobacterium longum

王貴貞(Kuei-Chen Wang)

指導教授 ： 李宗璘

共同指導教授 ： 林俊宏(Chun-Hung Lin)

國立臺灣大學/生命科學院/生化科學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.00133

摘要

比菲德氏菌(Bifidobacteria)為革蘭氏陽性厭氧菌，其具有獨特的lacto-N-biose I (LNB)/galacto-N-biose (GNB) 代謝路徑。比菲德氏菌利用此獨特的路徑代謝LNB及GNB，以利其共生在人類消化道中。LNB/GNB代謝路徑是由lnpABCD基因組經轉錄(transcription)、轉譯(translation)後生成。lnpA基因生成Galacto-N-biose/lacto-N-biose I磷酸化酶(galacto-N-biose/lacto-N-biose I phosphorylase, GLNBP)。 在不消耗ATP情況下，GLNBP可將LNB及GNB轉化為半乳糖-1-磷酸(galactose-1-phosphate) 及 氮-乙醯六碳糖胺 (N-acetyl-D-hexosamine)。氮-乙醯六碳糖胺包含氮-乙醯葡萄糖胺(N-acetylglucosamine, GlcNAc)及氮-乙醯半乳糖胺(N-acetylgalactosamine, GalNAc)。GLNBP有助於比菲德氏菌在厭氧環境中更有效運用ATP。接著，由lnpB基因生成的氮-乙醯六碳糖激酶 (N-acetylhexosamine 1-kinase, NahK)將氮-乙醯葡萄糖胺及氮-乙醯半乳糖胺磷酸化為氮-乙醯葡萄糖胺-1-磷酸(N-acetylglucosamine -1-phosphate, GlcNAc-1P)及氮-乙醯半乳糖胺-1-磷酸 (N-acetylgalactosamine-1-phosphate, GalNAc-1P)。NahK已被廣泛使用於酵素合成中。 雖然GLNBP及NahK已被研究多年，GLNBP及Nahk的分子催化機轉依然不清楚。因此，我們想利用X光蛋白結晶學及生物化學的方法解析出GLNBP及NahK的蛋白質結構，並進一步探討其催化作用機制。在此篇研究中，我們解析出GLNBP與LNB/GNB複合體蛋白質結構。藉由分析複合體蛋白結構，我們推測藉由無機磷酸根(inorganic phosphate)的直接攻擊，GLNBP可能是進行反轉的磷酸解反應(an inverting phosphorolytic reaction)。在NahK部分，我們解析了七個NahK與其受質(substrates)及產物(products)複合的蛋白結構。藉由這些複合體蛋白結構，我們推測NahK是進行類似SN2的磷酸化反應。NahK 是第一個具有多階段反應之複合體蛋白結構之激酶。GLNBP及NahK的蛋白複合體結構解析及催化機制的探討，將有助於GLNBP及NahK應用於寡糖的酵素合成並進一步增加寡糖的多樣性。

關鍵字

LNB ； GNB ； 磷酸解酶 ； 激酶

並列摘要

Bifidobacteria, gram-positive anaerobic bacteria, feature a unique lacto-N-biose I (LNB)/galacto-N-biose (GNB) pathway to utilize LNB and GNB for colonization in the gastrointestinal tract of humans. The LNB/GNB pathway is encoded in a four-gene lnpABCD operon. Galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) encoded by the gene lnpA catalyzes phosphorolysis of LNB and GNB into galactose-1-phosphate (Gal-1P) and N-acetyl-D-hexoamine (GlcNAc/GalNAc) without ATP consumption. This maximizes the effectiveness of ATP production under anaerobic conditions. A novel N-acetylhexosamine 1-kinase (NahK) encoded by the gene lnpB then phosphorylates N-acetyl-D-hexoamine to N-acetylhexoamine 1-phosphate (GlcNAc-1P/GalNAc-1P). NahK has been extensively used in chemoenzymatic synthesis of carbohydrates. However, the molecular mechanisms for both GLNBP and NahK remain unclear. In this study, we wanted to determine the crystal structures of GLNBP and NahK in complex with substrates/products to gain insight into the catalytic mechanisms of these two enzymes. For GLNBP, crystal structures of GLNBP in complex with LNB/GNB were solved. Based on the structural information, GLNBP was proposed to proceed through an inverting phosphorolytic reaction by the direct nucleophilic attack of an inorganic phosphate at the anomeric carbon. For NahK, seven 3-D structures of NahK in complex with GlcNAc, GalNAc, GlcNAc-1P, GlcNAc/AMPPNP and GlcNAc-1P/ADP were solved. Based on these snapshot structures, a direct in-line phosphoryl transfer mechanism was proposed. The NahK structures presented here represent the first multiple-reaction complexes of the enzyme. The demonstration of the protein crystal structures and the elucidation of the catalytic mechanisms would provide useful information for expanding the utilizations of both GLNBP and NahK to a great extent.

並列關鍵字

LNB ； GNB ； GLNBP ； NahK

參考文獻

1. Knight, D.J. & Girling, K.J. Gut flora in health and disease. Lancet 361, 1831 (2003).

2. Gibson, G.R. & Wang, X. Regulatory effects of bifidobacteria on the growth of other colonic bacteria. J Appl Bacteriol 77, 412-20 (1994).

3. Picard, C. et al. Review article: bifidobacteria as probiotic agents -- physiological effects and clinical benefits. Aliment Pharmacol Ther 22, 495-512 (2005).

4. Wei, M.Q. et al. Facultative or obligate anaerobic bacteria have the potential for multimodality therapy of solid tumours. Eur J Cancer 43, 490-6 (2007).

5. de Vrese, M. & Schrezenmeir, J. Probiotics, prebiotics, and synbiotics. Adv Biochem Eng Biotechnol 111, 1-66 (2008).