| Glycoside hydrolases were investigated from hyperthermophilic organisms to identify new hydrolases and determine biochemical similarity between families. Clan-D glycoside hydrolases (GH27 and GH36) were compared structurally and shown to share a conserved retaining reaction mechanism, catalytic residues, and structural features. Structural alignment and biochemical analysis of site-directed mutants of the family 36 alpha-galactosidase from Thermotoga maritima identified the catalytic residues as D327 and D387 for the nucleophile and acid-base residues, respectively. Azide rescue of D327G a concentration dependent increase in activity and Bronsted analysis showed a change in the rate-limiting step at pH 8-9. When the acid-base mutant, D387G, was rescued with the external anions azide and formate, a strong increase of activity was shown for galactoside substrates with a good leaving group (2,4-dinitrophenyl, DNP-Gal) over a poor leaving group (4-nitrophenyl, PNP-Gal). Similar increases were not noted for the wild-type enzyme. pH curves of the catalytic rate and efficiency for D387G showed no decrease in activity at higher pH.; A functional genomics approach, combined with bioinformatic techniques and biochemical characterization, was used to investigate the genome of the hyperthermophilic archaeon Pyrococcus furiosus to identify new glycoside hydrolases within the "hypothetical proteins". The open reading frame (ORF) PF0870 was identified by bioinformatic analysis to encode a glycoside hydrolase that hydrolyzes pNP-alpha-maltopyranoside and maltotriose, thereby defining it as a novel beta-amylase. Transcriptional response of P. furiosus grown on alpha-glucans suggested an alternative biochemical role for a previously characterized alpha-glucosidase. Purification and mass spectroscopy showed that the native alpha-glucosidase was encoded by "hypothetical protein" PF0132, which represents a new glycoside hydrolases family. Strong transcriptional response to growth on pullulan suggested activity on alpha-1,6-glucosidic linkages, and hydrolysis of the compounds maltose, isomaltose, panose, turanose, and maltotriose confirmed activity against this broad range of glucosidic oligosaccharides. This result demonstrates how functional genomics approaches, when combined with bioinformatics analysis and biochemical characterization, can be utilized to determine the role of proteins encoded in unannotated ORFs of genome sequences. |
