Phe427在Arthrobacter globiformis組織胺氧化酵素之受質特異性中的角色

Abstract

含銅胺類氧化酵素（Copper-containing amine oxidase, CAO）[EC1.4.3.6]廣泛存在於各種生物之中。該類酵素催化一級胺類的氧化脫胺作用且在各種不同生物有多元化的功能，如提供細菌生長所需的碳源及氮源、參與植物細胞防禦等機制，在動物中亦參與許多代謝機制。不同生物來源的含銅胺類氧化酵素對胺類受質特異性差別很大。Arthrobacter globiformis之組織胺氧化酵素（簡稱AGHO）屬於CAO的一種，為了了解哪些胺基酸決定AGHO的受質特異性，我們利用針對不同物種之CAO之間的序列比對及分子模擬方法選定了AGHO胺基酸序列中的Ala156、Pro157、Leu158及Phe427進行定點突變。兩套突變株分別模擬Hansenula Polymorpha 的甲基胺氧化酵素(methylamine oxidase，簡稱HPAO)及動物中牛血清胺類氧化酵素(Bovine serum amine oxidase，簡稱BSAO) 其活性區的胺基酸序列。酵素經由大腸桿菌表現、純化及活化步驟之後，測定突變株對於原本偏好的受質胺類與模擬之物種的受質胺類的活性及特異性。研究結果顯示模擬HPAO的突變株對於特定碳鏈長度的芳香族胺類選擇性提高；而模擬BSAO的突變株對原本偏好的受質之特異性降低，並對BSAO的偏好受質之特異性及活性提高。本研究說明了Ala156、Pro157、Leu158及Phe427這四個胺基酸位置扮演了與受質特異性有關的角色，並且證明針對特定胺基酸的定點突變可以改變AGHO之受質特異性。

Copper-containing amine oxidase (CAO) catalyzes the oxidative deamination of primary amines, and they are ubiquitous in nature and have diverse biological functions ranging from bacteria to mammals. CAOs from different sources possess various substrate preferences. Arthrobacter globiformis histamine oxidase (AGHO) is a member of the CAO family. To understand what residues in the active site determine the substrate specificity of AGHO, multiple sequences alignment and molecular modeling between different sources of CAOs were performed to pick four potential residues Ala156, Pro157, Leu158 and Phe427 for site-directed mutagenesis. Two sets of AGHO mutants were generated with one set mimics the active site residues of Hansenula Polymorpha methylamine oxidase (HPAO), while the other set mimics the active site residues of Bovine serum amine oxidase (BSAO). These mutants and the wild type AGHO were bacterial expressed, purified and subjected to activity and kinetics studies. In the report, the mutants mimicking HPAO altered the substrate specificity to aromatic amines with different chain lengths. Whereas, the mutants mimicking BSAO showed decreased specificities to histamine but exhibited a higher catalytic activity to typical substrates for BSAO. It is suggested that the Ala156, Pro157, Leu158 and Phe427 play important roles in the substrate recognition and selection. And the substrate specificity of AGHO could be modified or altered through site-directed mutagenesis.