Arthrobacter globiformis 胺類氧化酵素的表現,定點突變及特性之研究

Abstract

含銅胺類氧化酵素 [EC 1.4.3.6]普遍存於原核及真核生物，此酵素作用為催化生物胺類化合物之氧化而產生醛(aldehydes)、氨(ammonia)及過氧化氫(hydrogen peroxide)。 在原核生物中胺類氧化酵素提供了氮源及碳源的來源 ; 而在真核生物中則與細胞分化、細胞生長、去毒素作用、傷口的處理、細胞的訊號有關，甚至可能對於細胞死亡亦有關聯。 大體而言，含銅胺類氧化酵素以同源雙體 (homodimer) 的形式存在，依照來源其每一單元體分子量為70,000至105,000 daltons之間，每一單元含有一個銅離子及以胺基酸tyrosine經過後轉譯修飾作用所產生的共價結合的TPQ輔因子。從革蘭氏陽性菌Arthrobacter globiformis分離出來的含銅胺類氧化酵素中，依照胺基酸的相似性及受質專一性的不同劃分為兩類，其一為以histamine為受質，命名為histaminase 或 AGHO，另外一種則為以 phenylethylamine為主要受質的，命名為phenylethylamine oxidase 或 AGPEO。 在本研究中，我們將帶有AGHO的全長基因架至pET30b(+) 上，建構成一個帶有His6-Tag及AGHO全長的融合蛋白質。我們將已經將pET30b(+)/AGHO轉殖成功送入大腸桿菌BL21(DE3)中表現，首先以37 ℃培養後，加入新鮮的LB 培養液，添加50 μM 及0.05 mM IPTG 後在25 ℃誘導8小時，純化方式則選用硫胺沉澱及親和性管柱層析法，在1 L的培養液中，所得到的重組AGHO酵素產率為34 mg。 我們發現重組AGHO酵素對於高於溫度30 ℃的環境敏感，將重組酵素分別置於45 ℃、60 ℃ 30分鐘後，比起30 ℃作用的酵素活性各減少了10 %與40 %。在AGHO/HRP耦合酵素活性裡，當我們投以histamine 受質時，反應最適當的pH值為 8.0至pH 9.0之間。經過酵素動力學研究後，所得到的結果以phenylethylamine為受質時Km為134 μM， Vmax 為8.66 nmole/min/μg protein，而以histamine為受質時Vmax 為0.02 nmole/min/μg protein，Km為10.94 μM。 此外，本研究的另一個方向為此酵素及突變株的受質專一性探討，胺基酸Y316位置位於受質通道的閘道處，因此對於不同種的胺類受質具有選擇性，而此位置亦在反應中與TPQ輔因子的定位及去質子作用有關，因此我們將此胺基酸以點突變的方式改變成A、E、F、H、W。當我們投以不同種類的受質如 histamine, phenylethylamine, tyramine及 tryptamine ，結果顯示Y316位置的突變株具有比較低甚至沒有酵素的活性，這樣低的酵素活性據推測有可能與TPQ的形成有關。

Copper-containing amine oxidases [EC 1.4.3.6] present in both prokaryotes and eukaryotes. The enzymes use primary amines as substrates to catalyze the oxidative deamination and produce aldehydes, ammonia, and hydrogen peroxide. In prokaryotes, amine oxidases provide the source of carbon and nitrogen but in eukaryotes they are suggested to be involved in cell differentiation, cell growth, detoxification, wound healing, cell signaling, and possibly in cell death. Copper-containing amine oxidases are homodimers with molecular weight or single subunit ranging from 70,000 to 105,000 daltons depending on the sources. Each subunit of this type of enzymes contains a single copper ion and a covalently bound cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), formed by the post-translational modified tyrosine side chain. Two kinds of amine oxidases from gram positive bacterium Atrhrobacter globiformis have been identified based on their amino acid similarity and substrate specificity. One of two isoforms, termed histaminase or AGHO, prefers histamine as the substrate, while the other, phenylethylamine oxidase or AGPEO, prefers phenylethylamine as the major substrate. The AGHO gene encoding for histaminase has been subcloned from Atrhrobacter globiformis and inserted into an expression vector pET30(+) to give a fusion protein of full length AGHO and His6-tag. The His6-tagged protein was expressed in Escherichia coli strain BL21(DE3). The E. coli BL21(DE3) harboring pET30b(+)/ AGHO were grown at 37℃ in LB medium. Then, transfer to the fresh medium containing 50 μM CuSO4 and 0.05 mM IPTG at 25℃ for 8 hours. The purification was done by ammonium sulfate precipitation and affinity chromatography. The yield of recombinant AGHO was 33.75 mg/L cultural medium. The recombinant AGHO was found to be sensitive to temperature higher than 30 ºC. The enzyme activity decreased about 10 % and 40 %, when recombinant AGHO was kept at 45 ºC and 60 ºC, respectively, for 30 min. The optimal pH range for the AGHO/HRP coupled-enzyme activity assay in the presence of histamine and ABTS is between pH 8.0 and 9.0. The kinetic study showed that the Km for PEA is 134.9 μM and Vmaxis 8.66 nmole/min/μg protein.. The Km for histamine is 10.9 μM and Vmaxis 0.02 nmole/min/μg protein. Part of the goal of this research is to study the substrate specificities of wild type as well as AGHO mutants. The residue Y316 is found to locate at the gate of substrate channel and is postulated to play a role in recognition of various substrates. Y316 is also the residue that was shown to control the orientation of TPQ cofactor and to facilitate the deprotonation of TPQ during reaction. We have changed this residue into different amino acids, A, E, F, H, and W. We found that Y316 mutants showed low or no enzymic activity toward various substrates, including histamine, phenylethylamine, tyramine, and tryptamine. Interestingly, the low enzymic activity is due to the lack of the formation of TPQ in these Y316 mutants.