吩嗪類生物鹼分子生合成酵素之蛋白質表現、純化與試管內功能性探討

Abstract

吩嗪類生物鹼分子具有許多重要生物活性，如抗生素、抗癌、抗原蟲與抗瘧疾等。於本實驗室(Chiu Lab)先前的研究成果中，發現一段基因序列含有五個基因讀碼框，推測其功能可能為參與吩嗪類生物鹼分子或其中間產物之生物合成，分別為DAHPS、CHT、HDL、DHS和OXD。DAHPS為莽草酸生合成途徑的第一個酵素，推測其功能為催化磷酸烯醇丙酮酸(Phosphoenolpyruvate)和4-磷酸赤藓糖(erythrose-4-phosphate)而反應生成3-脫氧-D-阿拉伯庚酮醣-7-磷酸(3-deoxy-D-arabino-heptulosonate 7-phosphate)。CHT此酵素推測以分支酸(Chorismate)為受質，轉移胺基至分支酸上，並脫去分支酸上的氫氧基而形成鄰胺基脫氧異分支酸(Aminodeoxy-isochorismix acid)，接著HDL再以鄰胺基脫氧異分支酸為受質反應生成3-羫基-2-氨基苯甲酸(trans-2,3-dihydro-3-hydroxyanthranilic acid)。推測DHS其功能可能與3-羫基鄰胺基苯甲酸(3-hydroxyanthranilic acid)的生合成相關，OXD則是生合成吩嗪類生物鹼分子酵素之一。因此目標為鑑定與吩嗪類生物鹼分子生合成相關之酵素，進而建立其生合成途徑以完成產物的合成。本研究目前已經成功找到獲得此五個酵素的可溶性蛋白質的分析表現條件，亦成功異體表現並純化出CHT酵素，其酵素活性反應測試的結果，在經由高壓液相層析儀的分析後，成功地發現新的吸收峰。本研究吩嗪生物鹼分子生合成酵素之表達、純化與功能性鑑定，將有助於試管內吩嗪生合成途徑之建立。

Phenazine alkaloids have been found to play essential roles in mineral reduction and scavenging, transcriptional modulation and biocontrol, thereby displaying various biological properties such as antibiotic, antitumor, antimalaria and antiparasitic activities. For this study, a set of genes presumably involved in biosynthesis of yet-to-be identified metabolites, including DAHPS, CHT, HDL, DHS and OXD, have been previously proposed to function as DAHP synthase, aminodeoxyisochorismate synthase, isochorismatase, dehydrogenase and oxidase, respectively. Mechanistically, DAHPS could be involved in the first step of the shikimate pathway catalyzing the condensation of PEP and E4P to yield DAHP. Chorismate is considered to be a biosynthetic precursor first converted to ADIC by the enzyme CHT, followed by presumed catalytic action of HDL on ADIC to give DHHA. DHS could lead the biosynthesis pathway to 3-hydroxyanthranilic acid, and OXD could be involved in the synthesis of phenazine or novel compound. We aim to identify and characterize functions of the genes related to phenazine alkaloid biosynthesis. Developing an optimal process for heterologous gene expression and product analysis is an intended subject. In summary, I have succeeded to find the applicable expression condition of the five enzymes, DAHPS、CHT、HDL、DHS and OXD. And CHT has been purified to near homogeniety. Functional characterization of CHT is also described this study. The results provided a new opportunity for in vitro biosynthesis of phenazines and their analogs.