A differentially conserved residue (Ile42) of GH42 ß-galactosidase from Geobacillus stearothermophilus BgaB is involved in both catalysis and thermostability.

The glycoside hydrolase family 42 (GH42) of thermophilic microorganisms consists of thermostable ß-galactosidases that display significant variations in their temperature optima and stabilities. In this study, we compared the substrate binding modes of 2 GH42 ß-galactosidases, BgaB from Geobacillus stearothermophilus and A4-ß-Gal from Thermus thermophilus A4. The A4-ß-Gal has a catalytic triad (Glu312-Arg32-Glu35) with an extended hydrogen bond network that has not been observed in BgaB. In this study, we performed site-saturation mutagenesis of Ile42 in BgaB (equivalent to Glu312 in A4-ß-Gal) to study the effects of different residues on thermostability, catalytic function, and the extended hydrogen bond network. Our experimental results suggest that substitution of Ile42 with polar AA enhanced the thermostability but decreased the catalytic efficiency of BgaB. Polar AA substitution for Ile42 simultaneously affected thermostability, catalytic efficiency, and the hydrogen bond network, suggesting that Ile42 is responsible for functional discrimination between members of the GH42 family. These observations could lead to a novel strategy for investigating the functional evolution of the GH42 ß-galactosidases.