類黃酮磷酸酯合成酶結構分析及重要催化位點之研究

Abstract

類黃酮為一類自然界中普遍存在之植物多酚類二次代謝物，具備多方面豐富及重要之生理活性。然而文獻指出，大多數類黃酮化合物的水溶性不佳，生物可利用率(bioavailability)極低，使其應用受到很大的限制。本研究室先前篩選出 Bacillus subtilis BCRC 80517，可對daidzein及genistein 進行磷酸酯化修飾，形成daidzein 7-O-phosphate (D7P)及genistein 7-O-phosphate (G7P)，轉換後產物可大幅提升其水溶性及生物可利用率。先前研究室已自B. subtilis純化出該磷酸化酵素，完成蛋白質及基因定序，並成功利用大腸桿菌大量表現該酵素—類黃酮磷酸酯合成酶 (Flavonoid phosphate synthetase, FPS) 。本論文以此為基礎，進行後續研究。本研究首先探討FPS的同源蛋白質家族一級序列上之差異，並挑選出相同度 (identity) 約為70%、50%、25%之菌株，分別為Bacillus amyloliquefaciens BCRC 23350、Bacillus pumilus BCRC 11706及Bacillus megaterium BCRC 10608，透過四株菌對genistein生物轉換特性分析，我們確認同源蛋白質序列與BsnFPS之相同度在50%以下便不具有催化genistein之活性。另外，在SWISS-MODEL軟體的分析下，成功利用同源模擬法建立BsnFPS模擬三級結構，其substrate binding domain為十字架形狀，Apex為中軸、Arm1及Arm2為側臂，與swiveling domain包覆形成催化磷酸化反應之活性區。透過模擬結構與模板之序列及結構比對，找出可能為重要催化位點之胺基酸，接著以定點突變法建立蛋白質突變株，搭配酵素活性分析進行驗證，結果顯示，Lys27及His795分別為ATP binding domain及swiveling domain之重要催化位點，而Asp627、His629、His630則為Substrate binding domain中穩定His795上磷酸根之重要催化位點，而Glu329及Lys378則可能為受質活化位中與genistein鍵結有關的位點。

Flavonoids are polyphenolic secondary metabolites that are ubiquitous in nature and possess numerous health benefits. Nevertheless, research indicated that flavonoids have low aqueous solubility and poor bioavailability, and therefore limit its use. Our previous study screened out Bacillus subtilis BCRC 80517, which could phosphorylate daidzein and genistein and form daidzein 7-O-phosphate (D7P) and genistein 7-O-phosphate (G7P). These phosphate conjugates may be greatly improved in water solubility and bioavailability. The BCRC 80517 can also biotransform most of the flavonoids. In addition, we have also purified the enzyme, identified its protein and DNA sequence, and overexpressed the recombinant protein by E. coli. Nevertheless, among the biotransformed products, only isoflavone-phosphate conjugates have been identified so far. Therefore, the objective of this research is focusing on the flavonoid phosphate synthetase (FPS), and studied on the structure analysis and catalytic sites of BsnFPS. In this thesis, the homologous proteins sequences of FPS family were aligned at first. Bacillus amyloliquefaciens BCRC 23350、Bacillus pumilus BCRC 11706, Bacillus megaterium BCRC 10608 which exist homologous proteins with about 70, 50, 25% identity to BsnFPS were choosen then. After the in vivo and in vitro experiments of genistein bioconversion, the results showed that homologous proteins which was below 50% identity with BsnFPS would not have genistein catalytic activity. Otherwise, although BsnFPS and LmRPH (EC 2.7.9.6) present only 20% identity, they still have very similar secondary structure arrangement. Using SWISS-MODEL as a tool and LmRPH as a template, the structure of BsnFPS was successfully modeled by homology modeling. Moreover, according to the alignment between BsnFPS model and its template, we have found some putative catalytic sites. The results were confirmed by site-directed mutagenesis and enzyme activity assay, showing that H795, D627, H629, H630 are the major residues which catalyze phosphorylation of BsnFPS and K27 involves the binding of the ATP. E329 and K378 may form hydrogen bonds with genistein to stabilize it.