以定位點突變法探討綠竹液泡型蔗糖轉化酶活性區之結構與功能

Abstract

蔗糖轉化酶 (β-fructofuranosidase, EC 3.2.1.26) 是高等植物中參與蔗糖代謝的重要酵素之一，藉由蔗糖轉化酶的作用，蔗糖可被水解成葡萄糖與果糖，提供植物細胞利用。綠竹筍 (Bambusa oldhamii) 中至少存在三種蔗糖轉化酶異構酶，本研究以含有野生液泡型蔗糖轉化酶及其突變株cDNA的表現質體，轉形至酵母菌Pichia pastoris X-33進行重組蛋白質的表現。在1%甲醇誘導24小時後，以硫酸銨分劃及金屬螯合層析法等步驟純化液泡型蔗糖轉化酶及其突變株，進行酵素之蛋白質結構與功能關係的研究。 液泡型蔗糖轉化酶 (rIT3) 可被化學修試劑WRK、NBS及IAA抑制，蔗糖可保護酵素降低NBS的抑制作用。利用定位點突變配合酵素動力學實驗發現，Asp-127、Arg-251、Asp-252位置的突變會造成rIT3活性喪失，顯示這些胺基酸在酵素功能上扮演重要的角色。Asp-127、Glu-308為酵素催化主要的胺基酸，Cys-309以及Trp-151與穩定中間過渡狀態及酵素結構相關。rIT3與W151F、W151L、E308Q與C309A的最適反應pH值為5.0，在0~30℃的環境下較安定。突變株對蔗糖以及具有β-果糖苷基的棉仔糖親和力皆下降。在蔗糖轉化酶結構研究方面，成功利用同源模擬法 (Homology modeling)，以菊苣fructan 1-exohydrolase IIa為模版，得到綠竹筍液泡型蔗糖轉化酶可能的立體結構。此外，也成功得到rIT3可能的結晶條件，可作為後續微調最佳蛋白質結晶條件之參考。

Invertase (β-fructofuranosidase, EC 3.2.1.26) is one of the major enzymes in the sucrose metabolism of higher plants. The enzyme catalyzes the conversion of sucrose to glucose and fructose for plant cell utilization. There are at least three invertase isozymes in green bamboo (Bambusa oldhamii). To study the structure-function relationships of invertase, the expression plasmids containing wild-type vacuolar invertase or mutants cDNA were transformed into yeast Pichia pastoris X-33 for expression of recombinant proteins. After induction with 1% methanol for 24 hours, the recombinant wild-type and mutant proteins were purified by ammonium sulfate fractionation and affinity chromatography homogeneity. The enzyme activity of recombinant vacuolar invertase (rIT3) could be inactivated to various extent by the chemical modifying reagents, N-ethyl-5- phenylisoxazolium-3’-sulfonate (WRK), N-bromosuccinimide (NBS) and iodoacetate (IAA). Sucrose could protect the enzyme against the inactivation by NBS. Mutations in Asp-127、Arg-251、Asp-252 abolished the rIT3 activity. These results suggested that they are important residues in invertase function. Asp-127 and Glu-308 play important roles in catalysis. Cys-309 and Trp-151 play important roles in transition state and structural stability. Wild-type rIT3、W151F、W151L、E308Q and C309A have a pH optimum of 5.0 and they are stable at 0~30℃. Mutants have lower affinity with sucrose and raffinose than wild-type rIT3. The predicted 3D structure of Bambusa oldhamii vacuolar invertase was setup by Homology modeling which was based on the model of Cichorium intybus fructan 1-exohydrolase IIa. The crystallization conditions of rIT3 were also tested successfully, which could be used to find out the best condition of rIT3 crystal.