甘藷塊根質體型澱粉磷解酶構造中L78片段在不需醣引子之澱粉合成反應具有重要角色

Abstract

甘藷塊根中的質體型澱粉磷解酶 (L-form starch phosphorylase, Pho1) 具有一段78個胺基酸的序列，稱為L78。當L78存在時，Pho1可以在不含短鏈寡糖作為引子的狀態之下進行直鏈醣的合成，意即具有不需醣引子澱粉合成活性 (primer-independent activity, PI activity)。在前人的研究中，已知Pho1透過pyridoxal phosphate (PLP) 與第一個Glc-1-P結合，而L78上有三組Glu-Lys，可能組成Glc-1-P第二結合位。在本研究中以實驗室原有之L78表現蛋白為模板，製備6株在這三組Glu或Lys單點突變的L78 mutant，並將其添加回L78完整斷裂的Pho1*，發現Pho1可能主要透過Lys529與Glc-1-P結合。另外，在Pho1之L78的功能相關的研究方面，幾乎都會使用中央斷裂型Pho1 (Pho1*) 來觀察L78斷裂前後對其活性的影響。先前Pho1*主要是採用自然斷裂法來製備，但是缺點是花費時間久又無法調控。本論文嘗試以三種方法來促進Pho1的斷裂，以期能發展出中央斷裂型Pho1的標準化製備法。但三種方法皆無法成功的移除L78。未來可以構築具有人造蛋白酶切位的Pho1的表現載體，並使用特定的蛋白酶來移除L78。

L-form Starch phosphorylase (Pho1) from sweet potato contains a 78 amino acids residues insertion (Loop 78, L78) and may catalyze primer-independent activity (PI activity) to synthesize starch. Pho1 may lose its PI activity completely when L78 is degraded, and the addition of extrinsic expressed L78 rescues the PI activity of the degraded Pho1. Previous studies have indicated that the first Glc-1-P may be bounded to the active site of Pho1 via pyridoxal phosphate (PLP), while the second Glc-1-P binding pocket may compose of 3 pairs of Glu-Lys repeats including Glu528-Lys529, Glu534-Lys535 and Glu540-Lys541 in L78. In this study, we constructed expression vector for L78 as well as its mutants for Glu-Lys pairs and tested their effects on the PI activity. The results showed that mutation at Lys529 may block the PI activity, demonstrating that Lys529 could be a molecular determinant for the second Glc-1-P binding site. On the other hand, the studies for the role of L78 involve in the preparation of L78-removed Pho1 (Pho1*). Previous methods for Pho1* preparation are usually time-costing, and the quality of Pho1* lacks for consistency. This study used trypsin, heat treatment and proteasome to accelerate degradation of Pho1, respectively. But neither of them removed L78 successfully. In the future, we will construct Pho1 expression vector with artificial protease cutting site and use designated protease to remove L78.