PDBsum entry 5okb

Hydrolase

PDB id: 5okb

Contents

Protein chains

475 a.a.

Ligands

IMD ×8

GOL ×2

PO4 ×4

Waters ×2381

PDB id:

5okb

Name:

Hydrolase

Title:

High resolution structure of native gan1d, a putative 6-phospho-beta- galactosidase from geobacillus stearothermophilus

Structure:

Putative 6-phospho-beta-galactobiosidase. Chain: a, b, c, d. Engineered: yes

Source:

Geobacillus stearothermophilus. Organism_taxid: 1422. Gene: gan1d. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.33Å R-factor: 0.122 R-free: 0.145

Authors:

S.Lansky,A.Zehavi,Y.Shoham,G.Shoham

Key ref:

S.Lansky et al. (2017). Structural basis for enzyme bifunctionality - the case of Gan1D from Geobacillus stearothermophilus. FEBS J, 284, 3931-3953. PubMed id: 28975708 DOI: 10.1111/febs.14283

Date:

25-Jul-17 Release date: 18-Oct-17

Supersedes:

4zeh

Protein chains

W8QF82  (W8QF82_GEOSE) -  Putative 6-phospho-beta-galactobiosidase from Geobacillus stearothermophilus

Seq: 478 a.a.

Struc: 475 a.a.

Enzyme reactions

• Enzyme class: E.C.3.2.1.85 - 6-phospho-beta-galactosidase.
• Reaction: a 6-phospho-beta-D-galactoside + H2O = D-galactose 6-phosphate + an alcohol

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1111/febs.14283

FEBS J 284:3931-3953 (2017)

PubMed id: 28975708

Structural basis for enzyme bifunctionality - the case of Gan1D from Geobacillus stearothermophilus.

S.Lansky, A.Zehavi, H.Belrhali, Y.Shoham, G.Shoham.

ABSTRACT

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)₈TIM-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.