甘藷澱粉磷解酶重組蛋白之表現與活性分析

Abstract

Glycogen phosphorylase (GP) 的結構與功能已被詳盡研究，且證實在其N端有glucan結合區，而C端有Glc-1-P結合區以及pyridoxal phosphate (PLP) 結合區。依據GP的構造，可由電腦模擬 starch phosphorylase (SP) 的構形，除了分子中央多出一段 L78 loop，兩者構形十分相似。從甘藷中純化出來的 SP很容易被蛋白酶攻擊而斷裂，雖然斷裂的SP對整體構型及活性無太大影響，但只有完整SP存在時，才能顯現不需醣引子之合成澱粉活性 (primer independent activity, PI activity)；一旦L78斷裂後則會失去PI activity。為了更深入研究PI activity與SP結構之間的關係，我們利用基因重組建構全長以及不同長度之SP。N端部分經定序後發現有frame shift現象，SP的真正起始密碼，應該在原始位置 (ATG86) 的上游 (ATG52)。如此重新與馬鈴薯 SP 胺基酸序列進行比對，其相似性較原始序列高，約 83.6％。目前已植入保存載體有：全長SP、N端、C端、不含L78之N端與L78，但是成功表現出來的只有不包含L78之C端片段 (SPC)。此SPC片段上帶有catalytic domain與PLP binding domain，為His-tag融合蛋白，可用Ni-NTA純化。表現出來的SPC分離得可溶性與不可溶性兩部份，這兩類SPC都可被專一性抗體H7C所辨認。SPC能夠催化Glc-1-P釋出磷酸，但我們無法從活性染色看出有澱粉累積的現象。然而，SPC並非以磷酸酶的機制水解Glc-1-P而釋出Pi。雖然GP中PLP負責催化磷解與合成方向的機制已被證實，但仍未被證實於SP。SPC活性會隨PLP濃度增加而增加，表示PLP binding domain上的Lys 811可能接上PLP而參與催化。也運用圓二次光譜 (CD spectra) 與螢光光譜 (fluorescence spectra) 來確定PLP確實接上PLP binding site。針對PLP可能結合的Lys811位置進行點突變，目前已獲得一株突變株，但其活性不比預期低，需要進一步確認。

It was reported that glycogen phosphorylase has a glucan binding site in its N-terminal domain, while a Glc-1-P binding site and a pyridoxal phosphate (PLP) binding site in the C-domain. Starch phosphorylase (SP) has high structural homology to GP (38%) except that a loop of 78 amino acid residues (L78) is inserted in the middle of the molecule. It is difficult to purify the intact form of SP from sweet potato roots, since the L78 is very sensitive to proteolytic modification. We have found that, only the intact SP had the primer-independent activity (PI activity) for the synthesis of amylose from Glc-1-P. In order to investigate the mechanism of the PI activity and the structure-function relationship of SP, the expression of recombinant SP in both full-length and truncated forms is needed. The cloned sequence of the N-terminal peptide was not as what we have predicted, and the new initiation codon shifted 34 bases upstream from the published ATG. However, its sequence homology with the potato SP increased to 83.6%. We then tried to express the full-length and truncated forms of SP in E. coli system, only the C-terminal domain containing the catalytic site and PLP binding site was successfully expressed. This recombinant C-terminal peptide (SPC) is a His-tag fusion protein, which can be purified by Ni-NTA. We have isolated the soluble and the insoluble forms of SPC, both identified by the specific antibody H7C against the C-terminal part of SP. SPC showed the activity of releasing Pi from Glc-1-P. The amylose synthesizing activity can not be observed by activity staining on the gel. In addition, SPC showed no phosphatase activity. However, the phosphate-releasing activity of SPC was proportional to the addition of pyridoxal phosphate (PLP). PLP bound to SPC was analyzed by the fluorescence spectra and the circular dichroism spectra. We have constructed a PLP binding site mutant by replacing the Lys 811 to Ala. However, the activity of the mutant didn’t decrease as predicted.