Uncultured Neocallimastigales β-1,4-D-木聚醣水解酶的動力學分析及結構模擬

Kinetic studies and structural modeling for Uncultured Neocallimastigales β-1,4-D-xylanase

劉玟燕(Wen-Yan Liu)

指導教授 ： 蔡麗珠

國立臺北科技大學/工程學院/有機高分子研究所/碩士(2012年)

摘要

Uncultured Neocallimastigales 1,4-β-D-木聚醣水解酶(xynR8, EC 3.2.1.8)能催化水解木聚醣中β-1,4鍵結的醣苷鍵。我們觀察到此株xynR8與其突變株(N41D、N58D、N41D+N58D)皆有自然降解的現象，分子量從35kDa變成27kDa。酵素動力學結果顯示截短型的TxynR8和突變種(TN41D、TN58D、TN41D+TN58D)酵素對山毛櫸木中木聚醣的水解能力和效率都比原生種更佳。其中以TxynR8對山毛櫸木中木聚醣的活性最好，與受質間的比活性(specific activity)為11752 ± 324 units mg-1，而酵素的催化效率及專一性常數為kcat/Km為 1371 ml s-1 mg-1。我們也用Blast胺基酸序列比對找到TxynR8與晶體結構(PDB: 2VGD)有89%相似度，並用Modeler模擬TxynR8的結構，也將(PDB: 2VGD與1H4H)中類似受質分子放入模擬的TxynR8活性區域來觀察酵素活性區域的胺基酸與受質分子的鍵結關係，並得知突變位置Asn41 和 Asn58是扮演穩定結構和N端的loop，而Asn41也可能參與受質鍵結。而扮演催化水解反應的主要胺基酸是Glu144 和Glu232。

關鍵字

1,4-β-D-木聚醣水解酶突變株 ； 模擬 ； 酵素動力學

並列摘要

Uncultured Neocallimastigales 1,4-β-D-xylanase can hydrolyze and cleave β-1,4-glycosidic bonds in xylan. We found that 35 kDa xynR8 and its mutants (N41D、N58D、N41D+N58D) could degrade and the molecular weight became 27 kDa. The kinetic data showed that the truncated forms (TxynR8、TN41D、TN58D、 TN41D+TN58D) had more activity than that of wild type. TxynR8 had highest specific activity of 11752 ± 324 units mg-1 and the enzyme efficiency kcat/Km of 1371 ml s-1 mg-1 among all enzymes. In addition, we used Blast sequence alignment and Modeler to generate a TxynR8 structure based on the crystal structure (PDB:2VG9) which has 89% residue identity with TxynR8. The substrate-like (ferulic acid-1,5-arabinofuranose-α1,3-xylotriose) from PDB: 2VGD and (β-D-Xylopyranose) from PDB:1H4H were put into the active-site of xynR8 for enzyme-substrate interaction studied. Structural modeling suggests that residues Asn41 and Asn58 were required to stabilize the N-terminal loops and protein structure, and the Asn41 involved in substrate binding. The residues Glu144 and Glu232 play the important role in catalytic mechanism.

並列關鍵字

1,4-β-D-xylanase ； mutant ； model ； kinetic

參考文獻

1. Collins, T., Gerday, C., and Feller, G. (2005). Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiology Reviews 29, 3-23.

2. MacKenzie, L.F., Sulzenbacher, G., Divne, C., Jones, T.A., W?ldike, H.F., Sch?lein, M., Withers, S.G., and Davies, G.J. (1998). Crystal structure of the family 7 endoglucanase I (Cel7B) from Humicola insolens at 2.2 A resolution and identification of the catalytic nucleophile by trapping of the covalent glycosyl-enzyme intermediate. Biochem. J. 335, 409-416.

3. Davies, G., and Henrissat, B. (1995). Structures and mechanisms of glycosyl hydrolases. Structure (London, England : 1993) 3, 853-859.

4. Min, K.C. (2003). Crystal structure of human α-tocopherol transfer protein bound to its ligand: Implications for ataxia with vitamin E deficiency. Proceedings of the National Academy of Sciences 100, 14713-14718.

5. Vardakou, M., Dumon, C., Murray, J.W., Christakopoulos, P., Weiner, D.P., Juge, N., Lewis, R.J., Gilbert, H.J., and Flint, J.E. (2008). Understanding the structural basis for substrate and inhibitor recognition in eukaryotic GH11 xylanases. Journal of Molecular Biology 375, 1293-1305.