β-1,4-Glycanases of <i>Cellulomonas fimi:</i> Families, Mechanisms, and Kinetics

Bray, Mark R.; Creagh, A. Louise; Damude, Howard G.; Gilkes, Neil R.; Haynes, Charles A.; Jervis, Eric; Kilburn, Doug; MacLeod, Alasdair M.; Meinke, Andreas; Miller, Robert C.; Rose, David R.; Shen, Hua; Tomme, Peter; Tull, Dedreia; White, André; Withers, Stephen G.; Warren, R. A. J.

doi:10.1021/bk-1996-0655.ch006

Cited by 6 publications

(13 citation statements)

References 41 publications

(69 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…4A). This is consistent with the fact that all known xylanases are retainers, whereas many cellulases are inverters (25). Indeed, many of the known C. fimi cellulases are inverting glycosidases (25), which operate via a single-displacement mechanism and lack a covalent glycosyl-enzyme intermediate (22).…”

Section: Discussionsupporting

confidence: 80%

“…Identification of a Previously Undiscovered GH Family 10 Glycosidase in the C. fimi Secreted Proteome-The xylanolytic/cellulolytic soil bacterium C. fimi, which produces and secretes a complex array of xylanases and cellulases (25,26), is an ideal system for the discovery of new retaining ␤-1,4-glycanases. To date, four such C. fimi retaining enzymes (TABLE ONE) have been cloned and characterized (21,25,(27)(28)(29)(30). However, given the nature of the genetic cloning strategies, it is conceivable that some enzymes were missed (26).…”

Section: Discussionmentioning

confidence: 99%

“…the secreted proteome of the aerobic mesophilic xylanolytic/cellulolytic soil bacterium C. fimi. C. fimi secretes a complex array of xylanases and cellulases concurrently (25,26); however, only four C. fimi retaining ␤-1,4-glycanases have been characterized to date (see TABLE ONE) (21,25,(27)(28)(29)(30). Our MS-ABPP analysis, gene cloning, and kinetic characterization of the enzyme revealed that C. fimi secretes a previously unidentified family 10 glycanase with a carbohydrate-binding module (CBM) from CBM family 2b.…”

mentioning

confidence: 93%

See 2 more Smart Citations

Active-site Peptide “Fingerprinting” of Glycosidases in Complex Mixtures by Mass Spectrometry

Hekmat¹,

Kim²,

Williams³

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

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 ␤-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 ␤-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 ␤-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 TM accession number DQ146941) was cloned using inverse PCR techniques, and the protein was found to comprise a catalytic domain that shares ϳ70% 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.With the completion of the genome sequences of many organisms, the field of proteomics faces several major tasks. One challenge is that of identification and assignment of structure/function to the tens of thousands of proteins encoded by prokaryotic and eukaryotic genomes. Another challenge is accurate quantitative analysis of changes in protein levels/activities that occur within a proteome as a response to biological perturbations that are due either to normal developmental and metabolic changes or to abnormalities associated with disease. Proteomic techniques such as comparative two-dimensional gel electrophoresis coupled with mass spectrometry, the isotope-coded affinity tagging approach (1), and variations of isotope-coded affinity tagging that identify sites of modification (e.g. glycosylation and phosphorylation) on proteins (2-4) fail to provide a direct assessment of protein function.Recently, several chemical strategies for activity-based protein profiling (ABPP) 4 in complex proteomes that target several enzyme groups, including oxidoreductases (5, 6), serine hydrolases (7, 8), cysteine proteases (9, 10), threonine proteases (11), metalloproteases (12), protein phosphatases (13), kinases (14), and exoglycosidases (15), have been employed. ABPP probes have two general features: 1) an active sitedirected (mechanism-based) inactivator or affinity label that reacts with a catalytic residue and forms a covalent adduct with the target enzyme(s) and 2) one or more reporter groups that enable rapid detection (e.g. a fluorophore) and/or affinity isolation (e.g. biotin) (16). As such, ABPP methods can provide direct information on post-translational forms of protein regulation (17). However, most of the ABPP research ...

show abstract

Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 93%

See 1 more Smart Citation

Active-site Peptide “Fingerprinting” of Glycosidases in Complex Mixtures by Mass Spectrometry

Hekmat¹,

Kim²,

Williams³

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

Section: Analysis Of the C Fimi Secreted Proteomementioning

confidence: 99%

“…Like other xylanolytic/cellulolytic soil bacteria, C. fimi produces and secretes a complex array of xylanases and cellulases [29][30][31][32] of which there are four known retaining b-1,4-glycanases (Table 1). [21,29,[33][34][35] These enzymes are all modular proteins comprising from two to six modules or domains. [29,[32][33][34] They all have one or more catalytic domains (CD) and at least one carbohydrate-binding domain, the most common of which is the cellulose-binding domain (CBD).…”

Section: Analysis Of the C Fimi Secreted Proteomementioning

confidence: 99%

Synthesis and Testing of Mechanism‐Based Protein‐Profiling Probes for Retaining Endo‐glycosidases

2006

View full text Add to dashboard Cite

New functional proteomics methods are required for targeting and identification of subsets of a proteome in an activity‐based fashion. Glycosidases play critical roles in biology, yet a robust method for functional analysis of their activities and identities in biological proteomes is still lacking. An aryl 2‐deoxy‐2‐fluoro xylobioside inactivator was conjugated through cleavable and noncleavable linker arms to a biotin tag, thereby yielding two new active‐site‐directed reagents for activity‐based profiling of retaining β‐glycanases in complex proteomes. Crucially, these tagged reagents possess high specificity for their target enzymes with kinetic parameters similar to those of the untagged reagent. Western blotting showed that these reagents bind and covalently label active retaining β‐glycanases both in pure enzyme samples and in the secreted proteome of the soil bacterium Cellulomonas fimi. Such reagents therefore show great promise for future activity‐based targeting of glycanases.

show abstract

Specificity Fingerprinting of Retaining β‐1,4‐Glycanases in theCellulomonas fimiSecretome Using Two Fluorescent Mechanism‐Based Probes

et al. 2007

View full text Add to dashboard Cite

Functional proteomics methods are crucial for activity- and mechanism-based investigation of enzymes in biological systems at a post-translational stage. Glycosidases have central roles in cellular metabolism and its regulation, and their dysfunction can have detrimental effects. These enzymes also play key roles in biomass conversion. A functional profiling methodology was developed for direct, fluorescence-based, in-gel analysis of retaining beta-glycosidases. Two spectrally nonoverlapping fluorescent, mechanism-based probes containing different recognition elements for retaining cellulases and xylanases were prepared. The specificity-based covalent labelling of retaining glycanases by the two probes was demonstrated in model enzyme mixtures. Using the two probes and mass spectrometry, the secretomes of the biomass-converting bacterium Cellulomonas fimi, under induction by different polyglycan growth substrates, were analysed to obtain a specificity profile of the C. fimi retaining beta-glycanases. This is a facile strategy for the analysis of glycosidases produced by biomass-degrading organisms.

show abstract

β-1,4-Glycanases of Cellulomonas fimi: Families, Mechanisms, and Kinetics

Cited by 6 publications

References 41 publications

Active-site Peptide “Fingerprinting” of Glycosidases in Complex Mixtures by Mass Spectrometry

Active-site Peptide “Fingerprinting” of Glycosidases in Complex Mixtures by Mass Spectrometry

Synthesis and Testing of Mechanism‐Based Protein‐Profiling Probes for Retaining Endo‐glycosidases

Specificity Fingerprinting of Retaining β‐1,4‐Glycanases in theCellulomonas fimiSecretome Using Two Fluorescent Mechanism‐Based Probes

Contact Info

Product

Resources

About