6-磷酸-β-葡糖苷酶和6-磷酸-β-半乳糖苷酶是水解末端非还原葡萄糖-6-磷酸(Glc6P)或半乳糖-6-磷酸(Gal6P)之间的β-糖苷键的酶。分别,和其他有机分子。Gan1D,属于GH1家族的糖苷水解酶(GH),最近在嗜热脂肪土芽孢杆菌T-1细菌中的一个新特征的半乳聚糖利用基因簇中被鉴定。Gan1D已被证明具有双功能活性,同时具有6-磷酸-β-半乳糖苷酶和6-磷酸-β-葡萄糖苷酶活性。我们在这里报道了Gan1D的完整3D晶体结构,及其酸/碱催化突变体Gan1D-E170Q。Gan1D的三级结构与通常在GH酶中观察到的(β/α)8TIM桶折叠很好地相符,其四元结构采用二聚体组装,通过凝胶过滤和小角度X射线散射结果证实。我们还介绍了Gan1D的结构与推定的底物纤维二糖-6-磷酸(Cell6P)以及降解产物Glc6P和Gal6P的复合物。这些复合物揭示了Gan1D的特定酶-底物和酶-产物结合相互作用,以及与它的糖皮质激素有关的残留物,aglycone,和磷酸盐结合位点。我们表明,捕获在活性位点的不同配体采用不同的结合模式的蛋白质,为该酶观察到的双重半乳糖苷酶/葡糖苷酶活性提供了结构基础。根据这些信息,在一个活性位点残基(W433)上进行了特定的突变,将酶特异性从双重活性转变为对6-磷酸-β-葡萄糖苷酶活性的显着偏好。这些数据及其与相关葡糖苷酶和半乳糖苷酶的结构数据的比较用于对GH1酶的该亚组中的结构-功能关系的更一般的讨论。
Gan1D-野生型(WT)-P1,Gan1D-WT-C2,Gan1D-E170Q,Gan1D-WT-Gal6P,Gan1D-WT-Glc6P,和Gan1D-E170Q-Cell6P已保存在结构生物信息学研究实验室(RCSB)蛋白质数据库中,在加入代码5OKB下,5OKJ/5OKH,5OKA/5OK7,5OKQ/5OKK,5OKS/5OKR,和5OKG/5OKE,分别。
6-phospho-β-
glucosidases and 6-phospho-β-galactosidases are enzymes that hydrolyze the β-glycosidic bond between a terminal non-reducing glucose-6-phosphate (Glc6P) or galactose-6-phosphate (Gal6P), respectively, and other organic molecules. Gan1D, a glycoside hydrolase (GH) belonging to the GH1 family, has recently been identified in a newly characterized galactan-utilization gene cluster in the bacterium Geobacillus stearothermophilus T-1. Gan1D has been shown to exhibit bifunctional activity, possessing both 6-phospho-β-galactosidase and 6-phospho-β-glucosidase activities. We report herein the complete 3D crystal structure of Gan1D, together with its acid/base catalytic mutant Gan1D-E170Q. The tertiary structure of Gan1D conforms well to the (β/α)8 TIM-barrel fold commonly observed in GH enzymes, and its quaternary structure adopts a dimeric assembly, confirmed by gel-filtration and small-angle X-ray scattering results. We present also the structures of Gan1D in complex with the putative substrate cellobiose-6-phosphate (Cell6P) and the degradation products Glc6P and Gal6P. These complexes reveal the specific enzyme-substrate and enzyme-product binding interactions of Gan1D, and the residues involved in its glycone, aglycone, and phosphate binding sites. We show that the different ligands trapped in the active sites adopt different binding modes to the protein, providing a structural basis for the dual galactosidase/glucosidase activity observed for this enzyme. Based on this information, specific mutations were performed on one of the active site residues (W433), shifting the enzyme specificity from dual activity to a significant preference toward 6-phospho-β-glucosidase activity. These data and their comparison with structural data of related
glucosidases and galactosidases are used for a more general discussion on the structure-function relationships in this sub-group of GH1 enzymes.
Atomic coordinates of Gan1D-wild-type (WT)-P1, Gan1D-WT-C2, Gan1D-E170Q, Gan1D-WT-Gal6P, Gan1D-WT-Glc6P, and Gan1D-E170Q-Cell6P have been deposited in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank, under accession codes 5OKB, 5OKJ/5OKH, 5OKA/5OK7, 5OKQ/5OKK, 5OKS/5OKR, and 5OKG/5OKE, respectively.