title: Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases.
abstract: Organization of glycoside hydrolase (GH) families into clans expands the utility of information on catalytic mechanisms of member enzymes. This issue was examined for GH27 and GH36 through biochemical analysis of GH36 alpha-galactosidase from Thermotoga maritima (TmGalA). Catalytic residues in TmGalA were inferred through structural homology with GH27 members to facilitate design of site-directed mutants. Product analysis confirmed that the wild type (WT) acted with retention of anomeric stereochemistry, analogous to GH27 enzymes. Conserved acidic residues were confirmed through kinetic analysis of D327G and D387G mutant enzymes, azide rescue, and determination of azide rescue products. Mutation of Asp327 to Gly resulted in a mutant that had a 200-800-fold lower catalytic rate on aryl galactosides relative to the WT enzyme. Azide rescue experiments using the D327G enzyme showed a 30-fold higher catalytic rate compared to without azide. Addition of azide to the reaction resulted in formation of azide beta-d-galactopyranoside, confirming Asp327 as the nucleophilic residue. The Asp387Gly mutation was 1500-fold catalytically slower than the WT enzyme on p-nitrophenyl alpha-d-galactopyranoside. Analysis at different pH values produced a bell-shaped curve of the WT enzyme, but D387G exhibited higher activity with increasing pH. Catalyzed reactions with the D387G mutant in the presence of azide resulted in formation of azide alpha-d-galactopryanoside as the product of a retaining mechanism. These results confirm that Asp387 is the acid/base residue of TmGalA. Furthermore, they show that the biochemical characteristics of GH36 TmGalA are closely related to GH27 enzymes, confirming the mechanistic commonality of clan GH-D members.
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695-7905, USA.
authors.forename: Donald A
authors.initials: DA
authors.lastname: Comfort
authors.forename: Kirill S
authors.initials: KS
authors.lastname: Bobrov
authors.forename: Dina R
authors.initials: DR
authors.lastname: Ivanen
authors.forename: Konstantin A
authors.initials: KA
authors.lastname: Shabalin
authors.forename: James M
authors.initials: JM
authors.lastname: Harris
authors.forename: Anna A
authors.initials: AA
authors.lastname: Kulminskaya
authors.forename: Harry
authors.initials: H
authors.lastname: Brumer
authors.forename: Robert M
authors.initials: RM
authors.lastname: Kelly
mesh_terms: D000595:Amino Acid Sequence; D001386:Azides / Q000494:pharmacology; D002384:Catalysis; D003001:Cloning, Molecular; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D016415:Sequence Alignment; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics; D000519:alpha-Galactosidase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
chemical_list: D001386:Azides; D000519:alpha-Galactosidase