Site-directed mutagenesis to probe catalysis by a Thermobifida fusca beta-1,3-glucanase (Lam81A).
Thermobifida fuscaLam81A is a single domain family-81 beta-1,3-endoglucanase, but no structure is known for this family. Site-directed mutagenesis of 14 conserved residues chosen from sequence alignments was used to identify those with critical roles in catalysis, binding or substrate specificity. Mutant enzymes were assayed for their ability to bind and hydrolyze substrates with various glycosyl linkages. Residues D422, E499 and E503 were candidates for the catalytic acid or catalytic base, and E499 was shown to be the catalytic base by azide rescue. F425 was shown to have a major role in substrate binding possibly mediated by aromatic ring stacking with the sugar substrate. In addition, mutation of D424 to histidine altered the substrate specificity by increasing the rate of cleavage of mixed-linkage beta-glucan and carboxymethyl-cellulose, 60- and 16-fold, respectively, over the wild-type enzyme.
Department of Biochemistry, Vanderbilt School of Medicine, Nashville, TN 37232, USA.
Colleen E
CE
McGrath
Thu V
TV
Vuong
David B
DB
Wilson
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D055162:Biocatalysis; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D020134:Catalytic Domain; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D016415:Sequence Alignment; D019810:Sodium Azide / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D047071:beta-Glucans / Q000378:metabolism
D001426:Bacterial Proteins; D047071:beta-Glucans; D019810:Sodium Azide; D004693:Glucan Endo-1,3-beta-D-Glucosidase; D002266:Carboxymethylcellulose Sodium