Wiesmann C, Beste G, Hengstenberg W, Schulz G E
Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Freiburg, Germany.
Structure. 1995 Sep 15;3(9):961-8. doi: 10.1016/s0969-2126(01)00230-1.
The enzyme 6-phospho-beta-galactosidase hydrolyzes phospholactose, the product of a phosphor-enolpyruvate-dependent phosphotransferase system. It belongs to glycosidase family 1 and no structure has yet been published for a member of this family.
The crystal structure of 6-phospho-beta-galactosidase was determined at 2.3 A resolution by multiple isomorphous replacement, using the wild-type enzyme and a designed cysteine mutant. A second crystal form, found with the mutant enzyme, was solved by molecular replacement, yielding the conformation of two chain loops that are invisible in the first crystal form. The active center, located through catalytic residues identified in previous studies, cannot be accessed by the substrate if the two loops are in their defined conformation. The enzyme contains a (beta alpha)8 barrel and the relationship of its chain fold to that of other glycosidases has been quantified. As a side issue, we observed that a cysteine point mutant designed for X-ray analysis crystallized mainly as a symmetric dimer around an intermolecular disulfide bridge formed by the newly introduced cysteine.
The presented analysis provides a basis on which to model all other family 1 members and thereby will help in elucidating the catalytic mechanisms of these sequence-related enzymes. Moreover, this enzyme belongs to a superfamily of glycosidases sharing a (beta alpha)8 barrel with catalytic glutamates/aspartates at the ends of the fourth and the seventh strands of the beta barrel.
6-磷酸-β-半乳糖苷酶可水解磷酸乳糖，后者是磷酸烯醇丙酮酸依赖性磷酸转移酶系统的产物。它属于糖苷酶家族1，该家族成员的结构尚未见报道。
利用野生型酶和设计的半胱氨酸突变体，通过多同晶置换法在2.3埃分辨率下测定了6-磷酸-β-半乳糖苷酶的晶体结构。用突变体酶发现的第二种晶体形式通过分子置换法解析，得到了第一种晶体形式中不可见的两个链环的构象。如果这两个环处于其确定的构象，通过先前研究中鉴定的催化残基定位的活性中心无法被底物接近。该酶含有一个（β-α）8桶状结构，并且已对其链折叠与其他糖苷酶的链折叠之间的关系进行了量化。顺便提一下，我们观察到为X射线分析设计的半胱氨酸点突变体主要以对称二聚体形式结晶，围绕由新引入的半胱氨酸形成的分子间二硫键。
所提供的分析为模拟所有其他家族1成员提供了基础，从而将有助于阐明这些序列相关酶的催化机制。此外，该酶属于一个糖苷酶超家族，该超家族共享一个（β-α）8桶状结构，在β桶的第四和第七链末端具有催化性谷氨酸/天冬氨酸。
