利用點突變及X光結晶學分析Selenomonas ruminantium Phytase的活性區域

在植物的種子中含有大量的phytate (myo-inositol hexaphosphate，IP6) 來負責儲存磷酸 (Pi)，以利未來在發芽時所用。Phytase的作用為將phytate水解產生磷酸根及inositol polyphosphate衍生物。在畜牧業上的應用方面，也會將phytate及phytase作為飼料中的添加劑，以做為家禽或豬隻的磷酸來源。一般而言，phytase可分為Histidine acid phytase family及Alkaline phytase family兩種。而我們所要討論的Selenomous ruminantium phytase的作用機制並不屬於之前所了解的Histidine acid phytase family或Alkaline phytase family。藉由結構比對我們發現到其活性位置的結構高度相似於dual specificity phosphatase family。並且也發現到相較於其他物種的phytase，S. ruminantium phytase有著較高的催化能力。所以我們希望經由S. ruminantium phytase的研究來增加在商業上的應用價值。我們試著藉由點突變的方法，將主要負責催化的P-loop上Cys241改為Ser241、Ala241及有輔助弁鄋斡PD-loop上Asp212與Pro216分別改為Ala212與Gly216，來測量其在不同溫度下活性的改變，試著了解這些loop上這一些高度保留的胺基酸對催化活性的影響。發現到P216G的活性會隨著溫度的升高而下降。C241A、C241S、D212A則都會失去活性，這意味著這些位置跟催化弁鄏陬蛪奶j的關係。同時利用CD spectra，試圖由Tm值來檢視活性的改變是否是因為由於溫度的升高而使構型改變所造成。最後將C241A以E. coli大量表現，並以sodinm malonate為主沉澱劑來結晶，以X-ray的方式來了解phytase在結構上的特性及其與substrate的相互作用。由結構上卻意外的發現作為主沉澱劑的malonate會結合於活性位置。這個結構可以幫助我們了解polycarboxyl acid如何對phytase active site產生競爭抑制作用。

關鍵字

活性區域；點突變； X光結晶學；植酸

並列摘要

Phytase can hydrolyzes phytate（myo-inositol hexaphosphate）during plant germination to produce phosphate（Pi）and inositol polyphasphate derivatives. In poultry and pig farms, phytase and phytate are fed to livestock as additives to provide Pi. In this study, we analyzed the active site of Selenomous ruminantium phytase . It exhibits higher catalytic activity than many other phytases. We wish our analysis could improve its applicability in the livestock industry. Most phytases can be classified into two big families: Histidine acid phytase family and Alkaline phytase family. S. ruminantium phytase, however, belongs to neither of them. Through structural alignment we have found that the active site of S. ruminantium phytase greatly resembles to members of the dual specificity phosphatase family in that it contains conserved Cys241 in the primary catalytic site, P-loop, and Asp212 and Pro216 in the auxiliary site, WPD-loop. To study the functions of these conserved amino acid residues, we employed site-directed mutagenesis to change Cys241 into Ser241 or Ala241, and Asp212 and Pro216 into Ala212 and Gly216, respectively, and measured their catalytic activity of these mutant forms. We found that the activity of P216G decreases as the temperatures increases, whereas mutations in Cys241 and Asp212 abrogate the activity of S. ruminantium phytase. These results suggest that these sites are very important for the function of S. ruminantium phytase. We also examined CD spectra of these mutant forms to test whether the increase in temperature causes conformational changes. To better understand the structural properties of phytase and its interaction with the substrates, C241A recombinant protein is over expressed in E. coli, and crystallized by using sodium malonate as precipitant, and subject to X-ray crystallography. We found that malonate binds into the active site of C214A. This observation has gain insight that how the competitive inhibition of polycarboxyl acid acts to the phytase active site.

並列關鍵字

Active Site ； Site-directed Mutagenesis ； X-ray Crystallography ； Phytase

參考文獻

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利用點突變及X光結晶學分析Selenomonas ruminantium Phytase的活性區域

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