Active-site peptide "fingerprinting" of glycosidases in complex mixtures by mass spectrometry. Discovery of a novel retaining beta-1,4-glycanase in Cellulomonas fimi.
New proteomics methods are required for targeting and identification of subsets of a proteome in an activity-based fashion. Here, we report the first gel-free, mass spectrometry-based strategy for mechanism-based profiling of retaining beta-endoglycosidases in complex proteomes. Using a biotinylated, cleavable 2-deoxy-2-fluoroxylobioside inactivator, we have isolated and identified the active-site peptides of target retaining beta-1,4-glycanases in systems of increasing complexity: pure enzymes, artificial proteomes, and the secreted proteome of the aerobic mesophilic soil bacterium Cellulomonas fimi. The active-site peptide of a new C. fimi beta-1,4-glycanase was identified in this manner, and the peptide sequence, which includes the catalytic nucleophile, is highly conserved among glycosidase family 10 members. The glycanase gene (GenBank accession number DQ146941) was cloned using inverse PCR techniques, and the protein was found to comprise a catalytic domain that shares approximately 70% sequence identity with those of xylanases from Streptomyces sp. and a family 2b carbohydrate-binding module. The new glycanase hydrolyzes natural and artificial xylo-configured substrates more efficiently than their cello-configured counterparts. It has a pH dependence very similar to that of known C. fimi retaining glycanases.
Hekmat
Omid
O
Protein Engineering Network of Centres of Excellence of Canada, Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
Kim
Young-Wan
YW
Williams
Spencer J
SJ
He
Shouming
S
Withers
Stephen G
SG
D000595:Amino Acid Sequence; D001665:Binding Sites; D019921:Biotinylation; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry; D020134:Catalytic Domain; D040143:Cellulomonas / Q000201:enzymology*; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004187:Disaccharides / Q000037:antagonists & inhibitors; D006026:Glycoside Hydrolases / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D020543:Proteome / Q000737:chemistry; D040901:Proteomics / Q000379:methods; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013302:Streptomyces / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D002241:Carbohydrates; D004187:Disaccharides; D010455:Peptides; D011506:Proteins; D020543:Proteome; D006026:Glycoside Hydrolases; C020668:glycanase