醯亞胺水解酵素之序列分析與表現

Abstract

近年來，許多與醯亞胺水解酵素(dihydropyrimidinase；DHPase)有關的基因陸續被發現，它們的胺基酸序列與此酵素有50 ~ 60 %的高相同性(identity)，本實驗欲以分子演化(molecular evolution)的角度，應用生物資訊(bioinformatics)的方法，研究類似基因間彼此可能的關係，並設計實驗加以驗證。以BLAST軟體搜尋資料庫(library)內相同性高的蛋白質序列，經整理後歸為六大類。再分序列相似性及功能相似性兩主題，進一步分別比較其詳細之胺基酸，尋找、比對其序列上相似與相異之區域，由此來推測與蛋白質功能與胺基酸序列之相對關係。經比對後發覺位於靠近N端區域一段長約30個胺基酸的保留區域(conserved region) 推測可能與醯亞胺水解酵素的催化特性有相關。因此本實驗設計合成人類肝臟中的醯亞胺水解酵素 cDNA全長及缺乏N端約100個胺基酸的蛋白質並放入原核細胞內表現，以印證比對所得之推論。現階段表現出缺乏N端的蛋白，初步結果顯示，蛋白質形成了包含體(inclusion body)，經調整其生長及表現條件來改善其狀況；而所表現的產物並未測到活性。根據上述比對不同類型的蛋白質序列及實驗結果，初步推論N端之蛋白序列可能與DHPase活性有關。

Recently, many genes related to dihydropyrimidinase (DHPase) have been discovered and revealed 50 ~ 60 ﹪identity to DHPase. We used methods of bioinformatics to study the molecular evolution of these related proteins, and then designed experiment to study the structure/function relationship of DHPase and its related proteins. BLAST was used to search protein with high sequence identity ( > 40 %) with DHPase from DDBJ/EMBL/GenBank. The proteins were classified into six families by two approaches, through comparison of sequence homology and functionally related proteins. DHPase was compared with 8 sequence homologous proteins and 10 functionally related protein. We found that a region near N-terminal is conserved among functionally related proteins, but varied between DHPase and DHPase related proteins which do not have known catalytic function. We suggest that this region might be important for the catalytic function of DHPase. In order to investigate the significance of this region, we prepared and expressed an N terminal truncated protein in Escherichia coli. Conditions for the expression of the truncated DHPase were investigated to improve the production of soluble protein. No DHPase activity can be detected. Preliminary data indicates that N terminal of DHPase may be important for catalytic function.