Structure-function analysis of bacterial GH31 α-galactosidases specific for α-(1→4)-galactobiose.

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Glycoside hydrolase family 31 (GH31) encompasses alpha-glycoside hydrolases with diverse substrate specificities.
• GH31 has been recently classified into 20 subfamilies based on sequence similarity networks.
• An alpha-galactosidase from Bacteroides salyersiae (BsGH31_19) in subfamily 19 showed activity against synthetic substrates, but its natural substrate was unknown.

Purpose of the Study:

• To characterize BsGH31_19 and its ortholog FpGH31_19 from Flavihumibacter petaseus.
• To determine the substrate specificity and structural features of these GH31 alpha-galactosidases.
• To elucidate the molecular diversity and evolutionary relationships within GH31 and other alpha-galactosidase families.

Main Methods:

• Biochemical characterization of BsGH31_19 and FpGH31_19.
• Crystallographic analysis of the enzymes, including complex structures with substrates.
• Sequence and structural comparisons with known GH31 and other alpha-galactosidases.

Main Results:

• BsGH31_19 and FpGH31_19 exhibit high substrate specificity for alpha-(1->4)-linkages in alpha-(1->4)-galactobiose and globotriose.
• The enzymes form dimers with distinct interfaces compared to other GH31 members like PsGal31A.
• Structural analysis revealed variations in key amino acid residues involved in substrate recognition between BsGH31_19 and FpGH31_19, and homology of Trp153 to other alpha-galactosidases but not PsGal31A.

Conclusions:

• BsGH31_19 and FpGH31_19 possess unique substrate specificities, differing from previously characterized GH31 alpha-galactosidases.
• The structural data provides insights into the evolutionary divergence within GH31 subfamily 19 and across different alpha-galactosidase families.
• This study highlights the molecular diversity and evolutionary plasticity of glycoside hydrolases.