|標題:||以定點突變改變Arthrobacter globiformis 組織胺氧化酵素之受質特異性|
Alteration of Substrate Specificity of Arthrobacter globifomis Histamine Oxidase by Site-Directed Mutagenesis
|關鍵字:||組織胺氧化酵素;受質特異性;Arthrobacter globifomis Histamine Oxidase;Substrate Specificity|
|摘要:||含銅胺類氧化酵素 [EC220.127.116.11] 廣泛存在於細菌、黴菌、酵母菌、植物及動物界。此類酵素大都以同源雙體的形式存在，其中每一單體包含一個銅離子及一個共價結合的TPQ輔因子，並藉由氧化脫胺作用分解一級胺類進而產生醛類，氨及過氧化氫。然而不同來源的胺類氧化酵素彼此之間的受質特異性具有很大的差異。為了釐清其中的影響因素，我們利用不同來源的胺類氧化酵素胺基酸序列比對和QSAR 模型選出可能與受質特異性相關的胺基酸，分別是位在AGHO上A156、P157、L158這三個位置；之後依照HPAO (Hansenula Polymorpha metylamine oxidase)的序列，產生突變株A156D與A156D/L158W；且依照BSAO (Bovin serum amine oxidase)的序列，產生突變株A156S/P157G/L158D、A156S/P157G、以及L158D。實驗結果顯示所有的突變株皆具有正常的TPQ生成能力，但A156D與A156D/L158W的突變株具有較低甚至沒有酵素活性，推測原因可能是與Leu以Trp取代後造成的立體阻礙效應有關。而突變株A156S/P157G/L158D、A156S/P157G、及L158D對於aromatic amines的活性也是降低了；然而對於aliphatic amines的活性卻相對提升許多，尤其是BSAO的典型受質spermine。因此，在我們的研究中說明了A156、P157、以及L158可能扮演了與受質特異性有關的角色，並且成功地利用定點突變的方法改變了原有的受質特異性。|
Arthrobacter globiformis histamine oxidase (AGHO) is a member of copper-containing amine oxidase (CAO) family [E.C. 18.104.22.168], which is ubiquitous distributed in bacteria, fungi, plants and animals. CAOs generally catalyze the oxidation of various primary amines to their corresponding aldehydes, with the subsequent release of ammonia and hydrogen peroxide. CAOs contain a topaquinone (TPQ) in their active site as the redox cofactor. Substrate preference of CAOs varies; while AGHO exhibits a broad spectrum of substrate specificity to aromatic primary amines. To investigate the important factors influencing its substrate specificity, a multiple sequences alignment, molecular modeling and small molecule docking and site-directed mutagenesis were employed. Upon multiple amino acid sequences alignment and molecular modeling and small molecule docking, three resides in AGHO, termed A156, P157, and L158, were studied due to their potential roles in substrate recognition, structure stability, and enzyme activity. Accordingly, A156D, A156D/L158W (mimic the active site residues of HPAO (Hansenula Polymorpha metylamine oxidase)), A156S/P157G and A156S/P157G/L158D (mimic active site residues of BSAO (Bovin serum amine oxidase)) were generated, overexpressed and purified. We found the TPQ biogenesis was unaltered in these mutants. The catalytic activity of both A156D, A156D/L158W to aromatic amines was much lower than that of wild type, especially for A156D/L158W. It seems that, when Leu was changed to Trp, a steric effect may occur and hinder the access of substrate to the TPQ or cause an improper orientation of substrate directing to TPQ. Although, A156S/P157G/L158D, A156S/P157G, and L158D mutants also exhibit much lower catalytic activity to the aromatic amines than that of wild type AGHO, they show high catalytic activity to aliphatic amines, especially spermine, which is a typical substrate for BSAO. The results of this study suggest that A156, P157 and L158 play important roles in controlling the substrate specificity of AGHO and the replacement of these three amino acid residues may lead to conversion of substrate specificity of AGHO to the CAO from other species.
|Appears in Collections:||Thesis|
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