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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 分子生物學研究所 > 依資料類型分類 > 碩博士論文 >  Anabaena sp. CH1 N-acetyl-D-glucosamine 2-epimerase之生化及結構分析俾應用於酵素法生產N-acetyl-D-neuraminic acid

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/92943

標題: Anabaena sp. CH1 N-acetyl-D-glucosamine 2-epimerase之生化及結構分析俾應用於酵素法生產N-acetyl-D-neuraminic acid
Biochemical and Structural Study of Anabaena sp. CH1 N-Acetyl-D-Glucosamine 2-Epimerase for the Enzymatic Production of N-Acetyl-D-Neuraminic Acid
作者: 李晏忠
Lee, Yen-Chung
Contributors: 許文輝
中興大學
關鍵字: N-acetyl-D-neuraminic acid;anti-influenza virus agents;Whole Cell Catalyst;crystal structure;active sites;ATP-binding sites
唾液酸;抗流感藥物;全細胞生物觸媒;立體結構;活性中心;三磷酸腺苷;結合部位
日期: 2007
Issue Date: 2012-08-31 12:09:02 (UTC+8)
Publisher: 分子生物學研究所
摘要: 第一章
N-acetyl-D-neuraminic acid (NeuAc) 大量存在於細胞表面醣蛋白 (glycoproteins) 與醣脂類 (glycolipids) 的末端上,在細胞間辨識 (cell-cell recognition )、生物訊息傳遞(signal transduction)及致病菌感染等層面扮演著非常重要的角色。許多以 NeuAc 為基礎架構的藥物已被用來治療流行性感冒、糖尿病II型及癌症等多種疾病,為非常重要且昂貴的原料藥。以酵素法來生產 NeuAc 需利用 N-acetyl-D-glucosamine 2-epimerase (AGE)將 N-acetyl-D-glucosamine (GlcNAc) 轉換為 N-acetyl-D-mannosamine (ManNAc),再添加 pyruvate,由 NeuAc lyase 繼續催化合成 NeuAc。 為建立一個的高效能的全細胞生物催化系統,直接將基質 GlcNAc 及 pyruvate 轉換合成 NeuAc,本研究自藍綠藻 Anabaena sp. CH1 選殖 AGE 基因(bage),DNA 序列分析顯示 bage 長1,167 bp,轉譯出來的蛋白質為43 kDa(bAGE)。將 bage 與 E. coli NeuAc lyase 基因在 E. coli 大量表現,利用 Ni-NTA resin 親合層析方法純化出重組蛋白並進行生化性質分析。SDS-PAGE 分析顯示 bAGE 約有 20 % 為可溶性蛋白,而 NeuAc lyase 完全為可溶蛋白,bAGE 催化 GlcNAc 轉換為 ManNAc 之比活性為 124 U/mg,而 kcat 為 7.2 × 103 min-1。最適反應溫度為 45 ℃,在此溫度下之活性半衰期為 48 小時。 最適反應 pH 值為 8.0,在 pH 7 ~ 9.5 可維持 80 % 以上的活性。bAGE 在催化 GlcNAc 與 GlcNAc 間的 epimerization 需要 ATP 作為 activator ,非水解性 ATP 類似物 AMPPNP 以及 dATP 與 ADP 對 bAGE 具有與 ATP 相近之酵素活化作用。利用表現 bAGE 的 E. coli 為全細胞生物觸媒,催化 GlcNAc 轉換為 ManNAc 之比活性為 32 U/g cell;而以表現 E. coli NeuAc lyase 的全細胞生物觸媒,對催化 ManNAc 與 pyruvate 縮合成 NeuAc 之比活性為 132 U/g cell 。以 bAGE 搭配 E. coli NeuAc lyase 為全細胞生物觸媒可由 GlcNAc 與 pyruvate 直接催化合成 NeuAc,當 GlcNAc 與 pyruvate 皆為 1.2 M 時,GlcNAc 轉換率為 33.3 %,NeuAc 產率達到 10.2 g/L-h 的最高值,此時 NeuAc 的濃度為 0.4 M(123 g/L)。全細胞生物觸媒在循環使用 8 次後,GlcNAc 轉換率 及 NeuAc 產率仍能維持 80 % 以上。此全新的兩步全細胞生物觸媒合成 NeuAc 之製程,具有高產率、高觸媒循環使用性以及不需添加 ATP 等優點,具有工業化利用的潛力。
第二章
N-acetyl-D-glucosamine 2-epimerase (AGE) 催化 ManNAc 與 GlcNAc 間的 epimerization 為一可逆性的反應,目前對於此酵素參與催化反應之關鍵性殘基及其催化機制仍不瞭解。本研究首先由清大王雯靜教授實驗室利用 X 光繞射的方法以及 molecular replacement (MR) 的技術解出藍綠藻 Anabaena sp. AGE (bAGE)的3-D立體結構。其次,利用定點突變暨酵素動力學分析闡明 bAGE 活性中心內參與催化的關鍵性殘基。由解析度為 2.0 Å 的晶體結構發現 bAGE 為一雙元體構造 (dimer),每一單元體由十二個 α-helix(H1 ~ H12)組成 (α/α)6 barrel 的結構,是屬於 α/α toroid 的折疊方式。這些 α-helix 以一上一下的形式圍成桶狀的結構,中間形成一個很大的凹洞裂縫,此深深的凹洞狀似漏斗。裂縫開口一側的各 α-helix 之間有多個 b -sheet 及多個較長的 loop 構造,而在另一側則是由多個較短的 loop 連結各 α-helix。單元體 A 及 B 是以凹洞開口向外而背對背的形式靠在一起,雙元體是靠 H2-H3 內部與附近之殘基,以及 H8-H9 間的 short loops 以氫鍵與凡得瓦爾力交互作用而聚合。bAGE 與許多 glycoside hydrolases 以及醣代謝酵素一樣皆為 (α/α)6 barrel 的結構,屬於 Six-hairpin glycosyltransferases superfamily (GTs)。 針對 bAGE 結構中心凹洞內之保留性殘基 R57、E218、H239、E308、E242、H372 及 R375 進行點突變與動力學分析,發現 R57、H239、E308、H372 及 R375 等殘基對於催化正逆反應的活性都很重要。點突變酵素 R57A/K、E308A/D、H239A/N、及 H372A/I/N 皆失去正逆反應的活性,顯示這些殘基對酵素活性有關鍵性的影響。在不同 pH 測定 bAGE 之動力學顯示 bAGE 活性主要是在 pH 6-10 的範圍內。基於這些結果,我們假設一個 bAGE 的可互換式 deprotonation/reprotonation 機制。此外,分析突變酵素發現活性中心的殘基不影響酵素對 ATP 的結合,顯示 bAGE 的催化位置及 ATP 結合位置是分開的。
第三章
bAGE 在催化 GlcNAc 與 GlcNAc 間的 epimerization 可以利用 ATP、非水解性 ATP 類似物 AMPPNP、dATP 以及 ADP 作為 activator。為觀察 bAGE 對各種核苷酸之結合力,將核苷酸與 a-32P ATP 作競爭性結合並進行 UV cross-link,SDS-PAGE 及 X 光片放射顯影分析發現 dATP、AMPPNP 及 ADP 具有與 ATP 相當的強結合力;GTP、UTP 與 dGTP 次之,而 CTP、dCTP、dUTP 及 AMP 則最弱。ATP 的結合可保護 bAGE 不被 trypsin 水解,在沒有 ATP 存在下,bAGE 會被 trypsin 水解成 17 kDa 及 26 kDa 兩個片段。N 端定序與 MALDI-TOF MS 分析顯示 17 kDa 為第2殘基到 K155 之 N 端片段,26 kDa 為 Thr161 起始之 C 端片段。顯示在沒有 ATP 存在下,trypsin 會辨認 K155、K157 及 K160 三個殘基,而將 bAGE 水解成 N 端及 C 端兩個片段。推測 bAGE可能是以這些正電荷殘基與 ATP 作正負電荷作用而結合。分析定點突變的 bAGE 發現 K160A 與 I114R 變異蛋白幾乎尚失 ATP 結合力,顯示 K160 與 I114 兩殘基在參與 ATP 結合時扮演關鍵性的角色。在 1 mM ATP 存在下,K160A 及 I114R 變異蛋白分別只有野生酵素之 6 % 及5. 5 % 的活性,可能因 bAGE 無法結合 ATP 而不具有活性。推測 bAGE 之 K160 殘基可能與 ATP 之 phosphate 交互作用,而 I114 所在的 pocket 可能與 ATP 之 adenosine 交互作用。 此外,利用 In-gel digestion 與 MALDI-TOF MS 分析發現 bAGE 之 138-155 片段具有與 ATP 共價結合之殘基。此結果進一步確認 K160 及 I114 殘基在 ATP 與 bAGE結合上扮演關鍵性角色,同時也暗示 bAGE 可能與眾多 ATP 與 GTP 結合蛋白一樣,皆以一段 feasible loop 與 phosphate 進行結合。
Chapter I
N-acetyl-D-neuraminic acid (NeuAc) is an indispensable and most abundant amino sugar located at the end of a glycan chain in vetebrate glycoconjugates of cell surface. It plays a prominent role in numerous biological functions, including virus infection. Therefore, NeuAc could be a precursor for the manufacturing of many pharmaceutical drugs, such as anti-influenza virus agents. Zanamivir (Biota/Glaxo), a NeuAc derivative, that inhibits neuraminidases of influenza virus of both types A and B has been developed and commercially used to protect against the highly virulent H5N1 strain. To develop a whole cell process for Neu5Ac production, genes of Anabaena sp. CH1 N-acetyl-D-glucosamine 2-epimerase (bAGE) and Escherichia coli N-acetyl-D-neuraminic acid lyase (Neu5Ac lyase) was cloned and expressed in E. coli BL21 (DE3). The expressed bAGE was purified from the crude extract of IPTG-induced E. coli BL21 (DE3) (pET-bage) to homogeneity by nickel-chelate chromatography. The molecular mass of the purified bAGE was determined to be 42 kDa by SDS-PAGE. The pH and temperature optima of the recombinant AGE were pH 7.0 and 50 ºC, respectively, and only needs 20 mM ATP for maximal activity. The specific activity of bAGE (124 U/mg protein) for the conversion of N-acetyl-D-glucosamine to N-acetyl-D-manosamine was about 4-fold higher than that of porcine AGE. A stirred glass vessel containing transformed E. coli cells expressing age gene from Anabaena sp. CH1 and Neu5Ac lyase gene from E. coli NovaBlue separately was used for the conversion of GlcNAc and pyruvate to Neu5Ac. A maximal productivity of 10.2 g NeuAc/L.h with 33.3% conversion yield from GlcNAc could be obtained in a 12-h reaction. The recombinant E. coli cells can be reused for more than 8 cycles at a productivity of >8.0 g NeuAc/L.h. In this process, the expensive activator, ATP, necessary for maximal activity of porcine AGE in free enzyme system can be omitted.
Chapter II
N-acetyl-D-glucosamine 2-epimerase (AGE) catalyzes the reversible epimerization between N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-mannosamine (ManNAc). In collaboration with Dr. WC Wang's lab was determined the 2.0 Å resolution crystal structure of the AGE from Anabaena sp. CH1. The structure demonstrates an (α/α)6 barrel fold, which shows structural homology with porcine AGE as well as a number of glycoside hydrolase enzymes and other sugar-metabolizing enzymes. One side of the barrel structure consists of short loops involved in dimer interactions. The other side of the barrel structure is comprised of long loops containing six short beta-sheets, which enclose a putative central active-site pocket. Site-directed mutagenesis of conserved residues near the N-terminal region of the inner alpha helices shows that R57, H239, E308, and H372 are strictly required for activity. E242 and R375 are also essential in catalysis. Based on the structure and kinetic analysis, H239 and H372 may serve as the key active site acid/base catalysts. These results suggest that the (alpha/alpha)6 barrel represents a steady fold for presenting active-site residues in a cleft at the N-terminal ends of the inner alpha helices, thus forming a fine-tuned catalytic site in AGE. Nucleotide-binding and enzymatic activity assays of AGE variants also revealed that active sites and ATP binding sites of bAGE were structurally independent.
Chapter 3
Nucleotides, ATP, AMPPNP, dATP, and ADP can activate the epimerization activity of bAGE. To investigate the binding of nucleotides to bAGE, competitive binding of nucleotide and a-32P ATP to bAGE was evaluated by UV-mediated cross-linking and SDS-PAGE analysis. The results revealed that ATP, AMPPNP, ADP, and dATP had high affinitiy to the bAGE, while GTP, UTP, CTP, AMP, dGTP, dTTP, and dCTP showed weak or no binding affinity to the enzyme. ATP analog, AMPPNP, can also activate the bAGE activity indicated that hydrolysis of nucleotide is not involved in the catalytic reaction. The binding of ATP could protect the bAGE from trypsin digestion. In the absence of ATP, bAGE was digested by trypsin into two polypeptides with molecular masses of 17 kDa and 26 kDa. N-terminal amino acid sequencing and MALDI-TOF MS analysis demonstrated that three residues, K155, K157, and K160, situated in the feasible loop (residues 151-165) of bAGE were the targets for trypsin digestion, which can be protected by the binding of ATP. Site-directed mutagenesis of bAGE revealed that the K160 residue may be required for the interaction with the negative charge phosphate of ATP and residue I114 may interact with the adenosine part of ATP.
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