老鼠體內前列腺素還原脢之晶體結構及催化機制

Abstract

在老鼠體內纖維母細胞分化為脂肪細胞時，發現了15-酮基-前列腺素還原脢-2會大量表現，此酵素可將15-酮基-前列腺素E2作不可逆的降解作用，移除其高度的生物活性。 來自老鼠的15-酮基-前列腺素還原脢-2由351個氨基酸組成，分子量則為38015.3道爾頓，而此酵素已經在本實驗室成功地被選殖，並且在大腸桿菌中大量表現和純化。同時間，此酵素進行基本的活性測定。之後利用X光繞射原理成功地將15-酮基-前列腺素還原脢-2的酵素/受質共結晶結構解出來，老鼠的15-酮基-前列腺素還原脢-2屬於中鍊去氫脢/還原脢的其中一種。為了瞭解此酵素的催化機制，先經由和人體的15-酮基-前列腺素還原脢-2以及天竺鼠的白三烯酸B4去氫脢/15-酮基-前列腺素還原脢作比對之後，有幾個關鍵的殘基被選定出來，例如第64號位置的酪胺酸，第288號位置的息寧胺酸。利用定位突變的方式將這些殘基轉成側鍊類似的殘基，並利用活性測定觀察改變之後的結果。活性測定之後發現第64號位置的酪胺酸突變為苯丙胺酸之後活性下降了百分之九十五，而第288號位置的息寧胺酸如果突變為白胺酸之後反應活性下降了百分之四十，這些突變株和酵素與受質的共同晶體結構有助於推定並驗證老鼠的15-酮基-前列腺素還原脢-2的結構與功能相關性。

The mouse 15-keto-prostaglandin 13-reductase-2, mPGR2, was upregulated during the 3T3-L1 fibroblast differentiation and this enzyme plays a key role in the irreversible degradation of the highly biologically active 15-keto-prostaglandin E2 (15-keto-PGE2). The mPGR2 consists of 351 amino acids with molecular weight is 38015.3 Da. The mPGR2 cDNA has been cloned and expressed in E. coli system and purified to homogeneity. Enzymatic activity analysis of mPGR2 was completed and the ternary complex structure of mPGR2, NADP+, and 15-keto-PGE2 had also been solved at 2.2 A resolution by X-ray crystallography. The mPGR2 belongs to the medium-chain dehydrogenases/reductases (MDR) superfamily. Catalytic mechanism of mPGR2 was proposed preliminarily based on the structural alignment with human PGR2 (hPGR2) and guinea pig leukotriene B4 12-hydroxydehydrogenase/15-keto-prostaglandin reductase (gpLTB4 12-HD/PGR). Key residues, Y64 and T288 have been identified. Site-directed mutagenesis and activity assays of these mutants revealed that the Y64F and T288L mutants lost 95 % and 40 % catalytic activity, respectively. Together these results reveal structural and functional relationships of mPGR2.