Lansky Shifra, Zehavi Arie, Belrhali Hassan, Shoham Yuval, Shoham Gil
Institute of Chemistry, The Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Israel.
Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa, Israel.
FEBS J. 2017 Nov;284(22):3931-3953. doi: 10.1111/febs.14283. Epub 2017 Oct 23.
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) 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.
6-磷酸-β-葡萄糖苷酶和6-磷酸-β-半乳糖苷酶是分别水解末端非还原性6-磷酸葡萄糖（Glc6P）或6-磷酸半乳糖（Gal6P）与其他有机分子之间β-糖苷键的酶。Gan1D是一种属于GH1家族的糖苷水解酶（GH），最近在嗜热栖热放线菌T-1中新鉴定的一个利用半乳聚糖的基因簇中被发现。已证明Gan1D具有双功能活性，兼具6-磷酸-β-半乳糖苷酶和6-磷酸-β-葡萄糖苷酶活性。我们在此报告了Gan1D及其酸碱催化突变体Gan1D-E170Q的完整三维晶体结构。Gan1D的三级结构与GH酶中常见的（β/α）TIM桶状折叠结构非常吻合，其四级结构为二聚体组装，这已通过凝胶过滤和小角X射线散射结果得到证实。我们还展示了Gan1D与假定底物6-磷酸纤维二糖（Cell6P）以及降解产物Glc6P和Gal6P形成的复合物的结构。这些复合物揭示了Gan1D特定的酶-底物和酶-产物结合相互作用，以及参与其糖苷、苷元和磷酸结合位点的残基。我们表明，捕获在活性位点的不同配体与蛋白质采用不同的结合模式，为该酶观察到的双半乳糖苷酶/葡萄糖苷酶活性提供了结构基础。基于这些信息，对其中一个活性位点残基（W433）进行了特定突变，使酶的特异性从双功能活性转变为对6-磷酸-β-葡萄糖苷酶活性有显著偏好。这些数据及其与相关葡萄糖苷酶和半乳糖苷酶结构数据的比较，用于对GH1酶这一亚组的结构-功能关系进行更全面的讨论。
Gan1D野生型（WT）-P1、Gan1D-WT-C2、Gan1D-E170Q、Gan1D-WT-Gal6P、Gan1D-WT-Glc6P和Gan1D-E170Q-Cell6P的原子坐标已分别存入结构生物信息学研究合作实验室（RCSB）蛋白质数据库，登录号分别为5OKB、5OKJ/5OKH、5OKA/5OK7、5OKQ/5OKK、5OKS/5OKR和5OKG/5OKE。
