Reaction mechanism based on X-ray crystallography at atomic resolution of endopolygalacturonase

[目的]在大肠杆菌中表达、纯化和结晶紫草内切多聚半乳糖醛酸酶I，构建表达系统和纯化方法，制备具有功能活性的EndoPG I，用于突变和结晶学研究。而在BL21(DE3)中表达的酶定位于包涵体中。利用阳离子交换层析一步法分离纯化了来自紫胸菌的内切多聚半乳糖醛酸酶IVa和IVb的纯化、性质及氨基酸序列分析。为了分析内切多聚半乳糖醛酸酶I的功能，我们对等电点不同的紫胸菌内切多聚半乳糖醛酸酶IV进行了纯化、鉴定和氨基酸序列分析。通过三步柱层析，从培养滤液中纯化出两个EndoPG IV、VIA和VIB。ESI-MS测定EndoPG Via和VIB的相对分子质量分别为36,017 Da和37,266 Da。这些PG具有共同的一级结构，分别具有一个和两个糖链。通过克隆含有5‘-RACE和3’RACE的基因，确定了其氨基酸序列。在此EndoPG IV中，没有观察到成熟EndoPG I的C-末端(44个氨基酸残基)缺失。EndoPG IV与成熟EndoPG I的氨基酸序列有72%的同源性。在pH 2.5条件下，用X射线结晶学测定一般碱性催化。因此，我们尝试在pH值为2.5时用X-射线单晶衍射法测定一般碱性催化作用。结果表明，Asp153似乎是碱基催化剂候选者。

○Expresion, purification and crystallization of Sterum purpureum endopolygalacturonase I in Escherichia coli.We constructed an expression system, using Escherichia coli, and a purification method to prepare functionally active endoPG I for the mutation and crystallographic studies. Expression in E.coli strain Origami (DE3) provided soluble and active enzyme with proper disulfide bonds, whereas the enzyme expressed in BL21(DE3) was localized in inclusion bodies. Sufficient amount of recombinant endoPG I produced by Origami (DE3) was purified by a single-step procedure using cation exchange chromatography.○Purification, characterization and amino acid sequence of endopolygalacturonase IVa and IVb from Sterum purpureum.To analysisi function of endoplygalacrturonase I we traied purification, characterization and amino acid sequence analysis of endopolygalacturonase IV, which have difference isoelectoric point and is produced by the same fungus Stereum purpureum. Two EndoPG IV, VIa and VIb were purified to homogeneity by three steps of column chromatography from the culture filtrate. The molecular masses of the EndoPG VIa and VIb were determined to be 36,017Da and 37,266Da using ESI-MS, respectively. These PGs have the common primary structure and one and two of sugar chain, respectively. By cloning of the cDNA with 5'-race and 3'race, the ammo acid sequence was decided. In this EndoPG IV, there are no deletion of C-terminal region (44 amino acid residues) observed in mature EndoPG I. The EndoPG IV was 72 % homology for amino acid sequences of mature EndoPG I.○Determination of general basic catalysis by X ray crystallography at pH 2.5.The pKa_1 and pKa_2 of the enzyme were determined at 4.35 and 5.3, respectively. Therefore, we tried to determine general basic catalysis by Xray crystallography at pH2.5 by for low. As the results, Asp153 seems to be the base catalyst candidate.