人類醯亞胺水解酵素之分子選殖表現及其功能性質探討

Abstract

醯亞胺水解酵素(DHP, dihydropyrimidinase, EC 3.5.2.2 )參與嘧啶的還原性分解途徑。人類醯亞胺水解酵素基因座落於染色體位置8q22，含1560個鹼基所構成的開放讀碼框，編碼著由519個胺基酸所構成的多肽鏈。人類醯亞胺水解酵素屬於cyclic amidohydrolase superfamily，其中包含了dihydropyrimidase、allantoinase、hydatoinase、dihydroorotase等，皆參與嘌呤與嘧啶環的代謝。分析人類醯亞胺水解酵素的胺基酸序列，其與大鼠及小鼠分別有90%及88%的相似度。人類醯亞胺水解酵素缺乏症是一種體染色體隱性遺傳的疾病，不但可藉由檢測病患尿液中dihydropyrimidine含量來判斷是否患有醯亞胺水解酵素缺乏症，而且與許多臨床病徵如癲癇、畸形、心理及生理發育不完全有關。然而截至目前為止，在蛋白質層面，人類醯亞胺水解酵素仍有尚未被了解的生化特性及功能。為了分析人類醯亞胺水解酵素的特性，我們解決在大腸桿菌內表現人類醯亞胺水解酵素所遇到的蛋白質包涵體(Protein inclusion body)問題，在大腸桿菌內大量表現可溶並具活性的人類醯亞胺水解酵素，並進一步比較不同物種來源，在酵素動力學、pH值及溫度對活性的影響、耐熱度、耐鹽度上的差異。除此之外我們還積極地建立一個能穩定生產人類醯亞胺水解酵素的平台，以便後續能夠從蛋白質層面來探討，在病理上如何因為單一個氨基酸突變而造成人類醯亞胺水解酵素失去活性，導致人類醯亞胺水解酵素缺乏症的發生。

Imidase participates in the reductive degradation pathway of pyrimidine. The location of Homo sapiens dihydropyrimidinase (hDHP, DPYS, NM_001385) gene is on the chromosome 8q22 and its cDNA clone contains 1560-bp open reading frames encoding polypeptides of 519 residues. hDHP belongs to the superfamily of cyclic amidohydrolase, including dihydropyrimidase, allantoinase, hydatoinase, dihydroorotase, all of which are involved in the metabolism of purine and pyrimidine rings. The deduced amino acid sequence of hDHP shows 90% and 88% identity with that of rat and mouse DHP, respectively. hDHP deficiency is an automosomal recessive disorder characterized by dihydropyrimidinuria and associated with variable clinical phenotypes, such as seizures, mental retardation, growth retardation and dysmorphic features. However, little is known about the properties and biological functions of human imidase at protein level. In order to characterize the biochemical properties of human imidase, we expressed human imidase gene in E. coli and rendered the purified protein in soluble and active form. The characterizations in enzyme kinetics, the effect of pH and temperature to enzyme activity, thermostability and salt tolerance of human imidase and those from other species were compared. This provided a basis for the future preparation of imidase mutants to study the effect of single amino acid substitution on imidase that causes hDHP deficiency