GanB is a β-galactosidase that belongs to family 42 of glycoside hydrolases (GH42). The enzyme was isolated from the thermophilic bacterium Geobacillus stearothermophilus T-6 as a part of a gene cluster that encodes for six proteins involved with the utilization of β-1,4-galactan. GanB hydrolyzes short galacto-oligosaccharides with an exo mode of action but is not active on high Mw galactan (Tabachnikov & Shoham, FEBS J. 2013).The crystal structure of the nucleophile mutant of GanB, GanB-E323A, was determined at 2.5 Å resolution by molecular replacement, using the Thermus thermophilus A4 β-galactosidase A4-β-Gal (PDB code: 1kwk) as a search model. The quaternary structure of GanB is homotrimer, where each subunit is composed of three structural domains. The interactions between subunits form a pocket shaped active site, which is typical to exo-acting enzymes. Moreover, Trp197, that is located on a loop at the surface of each subunit, can form a hydrogen bond via a water molecule with galactose that is bound at the catalytic site of the next subunit, and thus participates directly in catalysis. A Zn+2 ion that is bound by four conserved cysteine residues creates a coordination complex which contributes to the thermal stability of the protein. Furthermore, this structural feature is important to the exact positioning of Trp197 in the active site, and therefore affects catalysis.
