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

Abstract

The glycoside hydrolase family 42 (GH42) of thermophilic microorganisms consists of thermostable β-galactosidases that display significant variations in their temperature optima and stabilities. In this study, we compared the substrate binding modes of 2 GH42 β-galactosidases, BgaB from Geobacillus stearothermophilus and A4-β-Gal from Thermus thermophilus A4. The A4-β-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-β-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 β-galactosidases.

Crystal structure of β1→6‐galactosidase from Bifidobacterium bifidum S17: trimeric architecture, molecular determinants of the enzymatic activity and its inhibition by α‐galactose

“…Similar to other β‐galactosidases from GH42 family, domain I contains the catalytic residues Glu160 (nucleophile) and Glu320 (acid/base). The overall RMSDs calculated for BbgII Cα atoms and the correspondent atoms of homologous Bca‐β‐gal , A4‐β‐Gal , GanB , and BlGal42A structures are 1.53 Å, 1.82 Å, 2.0 Å, and 0.6 Å, respectively. As expected, the structure of the catalytic domain I is more conserved, with the RMSDs calculated for 398 Cα atoms of 1.0 Å, 1.20 Å, 1.6 Å, and 0.53 Å for Bca‐β‐gal, A4‐β‐Gal, GanB, and BlGal42A structures, respectively.…”

“…Similar to other b-galactosidases from GH42 family, domain I contains the catalytic residues Glu160 (nucleophile) and Glu320 (acid/base). The overall RMSDs calculated for BbgII Ca atoms and the correspondent atoms of homologous Bca-b-gal [25], A4-b-Gal [24], GanB [26], and BlGal42A [27] The general aspect of the BbgII domain I is the same for all homologous structures of GH42 family members with about 400 residues, assembled in a TIM-barrel fold with eight a-helices and eight bstrands ( Fig. 1A).…”

“…The GH42 family has only four structurally characterized representatives: β‐galactosidases from Thermus thermophilus [A4‐β‐Gal, Protein Data Bank (PDB) id: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=1KWG] ; Bacillus circulans sp. alkalophilus (Bca‐β‐gal, PDB id: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3TTS) ; Geobacillus stearothermophilus (GanB, PDB id: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4OIF) ; and B. animalis subsp. lactis Bl‐04 (BlGal42A, PDB id: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4UNI) .…”

“…The GH42 family has only four structurally characterized representatives: b-galactosidases from Thermus thermophilus [A4-b-Gal, Protein Data Bank (PDB) id: 1KWG] [24]; Bacillus circulans sp. alkalophilus (Bcab-gal, PDB id: 3TTS) [25]; Geobacillus stearothermophilus (GanB, PDB id: 4OIF) [26]; and B. animalis subsp. lactis Bl-04 (BlGal42A, PDB id: 4UNI) [27].…”

β-galactosidase: Biotechnological applications in food processing

“…Substrate binding modes of 2 GH42 β‐galactosidases, Bga B from Geobacillus stearothermophilus , and A4‐β‐Gal from Thermus thermophilus A4 were compared with know the basis of the catalytic efficiency of the enzyme. The A4‐β‐Gal has a catalytic triad (Glu312‐Arg32‐Glu35) with an extended hydrogen bond network that has not been observed in BgaB (Dong, Hai‐Qin, Yan‐Hui, Hao, & Wei, ).…”