Cellulose-binding polypeptides from Cellulomonas fimi: endoglucanase D (CenD), a family A beta-1,4-glucanase

Meinke, Andreas; Gilkes, Neil R.; Kilburn, Douglas G.; Miller, Robert C.; Warren, R. A. J.

doi:10.1128/jb.175.7.1910-1918.1993

Cited by 61 publications

(61 citation statements)

References 38 publications

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“…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%

“…5). Of the other three known but undetected ␤-1,4-glycanases (TABLE ONE), CenD has extremely low xylanase activity and is not expected to react with DNP2FX 2 SSB (27), and XylC is likely intracellular because it lacks the leader sequence typical of secreted prokaryotic proteins (28). XylD is extracellular, but it is probably secreted in minute amounts (below the detection limit of the ABPP methodology) under the growth conditions employed.…”

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%

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

Hekmat¹,

Kim²,

Williams³

et al. 2005

Journal of Biological Chemistry

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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 ...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 93%

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

Hekmat¹,

Kim²,

Williams³

et al. 2005

Journal of Biological Chemistry

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“…However, whereas extremely useful as a fast screen this plate assay was still not quantitative, perhaps because of the unequal growth of cells on agar plates and the inefficient expression of Cel5A (ϳ0.5 mg/liter culture) compared with the glycosynthase (ϳ50 mg/liter culture) in the present expression system. The reason for such an unbalanced expression is unclear but low-level expression of cellulases from C. fimi in E. coli systems has previously been reported (20,39). To address these problems, a 96-well plate assay was designed for positive clones that had been regrown.…”

Section: Discussionmentioning

confidence: 99%

“…Construction of pGSVIII as a Screening Vector-The gene encoding the catalytic domain of cellulase D (Cel5A) from Cellulomonas fimi (20) was amplified by PCR using 1 M T7 promoter primer (5Ј-TAATACG-ACTCACTATAGGG) and the Cel5A-TERM primer (5Ј-CCCTCTAGAT-TAAAGCTTGACCTGCGAGATCGA), 0.2 mM each of the four dNTPs, 5% dimethyl sulfoxide, 25 ng of pGSVIICel5A (19), template DNA, and 2.5 units of Pwo polymerase (Roche Diagnostics) in 50 l of 1ϫ Pwo polymerase buffer. Twenty-five PCR cycles (45 s at 94, 30 s at 55, and 80 s at 72°C) were performed in a thermal cycler (PerkinElmer Life Sciences, GeneAmp PCR System 2400).…”

Section: Methodsmentioning

confidence: 99%

Directed Evolution of a Glycosynthase from Agrobacterium sp. Increases Its Catalytic Activity Dramatically and Expands Its Substrate Repertoire

Kim

Lee

Warren

et al. 2004

Journal of Biological Chemistry

122

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The Agrobacterium sp. ␤-glucosidase (Abg) is a retaining ␤-glycosidase and its nucleophile mutants, termed Abg glycosynthases, catalyze the formation of glycosidic bonds using ␣-glycosyl fluorides as donor sugars and various aryl glycosides as acceptor sugars. Two rounds of random mutagenesis were performed on the best glycosynthase to date (AbgE358G), and transformants were screened using an on-plate endocellulase coupled assay. Two highly active mutants were obtained, 1D12 (A19T, E358G) and 2F6 (A19T, E358G, Q248R, M407V) in the first and second rounds, respectively. Relative catalytic efficiencies (k cat /K m ) of 1:7:27 were determined for AbgE358G, 1D12, and 2F6, respectively, using ␣-D-galactopyranosyl fluoride and 4-nitrophenyl ␤-D-glucopyranoside as substrates. The 2F6 mutant is not only more efficient but also has an expanded repertoire of acceptable substrates. Analysis of a homology model structure of 2F6 indicated that the A19T and M407V mutations do not interact directly with substrates but exert their effects by changing the conformation of the active site. Much of the improvement associated with the A19T mutation seems to be caused by favorable interactions with the equatorial C2-hydroxyl group of the substrate. The alteration of torsional angles of Glu-411, Trp-412, and Trp-404, which are components of the aglycone (؉1) subsite, is an expected consequence of the A19T and M407V mutations based on the homology model structure of 2F6.Oligosaccharides have considerable potential as therapeutics because of the numerous medicinally relevant physiological events that involve glycoconjugates (1-3). To expand our understanding of the various roles of oligosaccharides found in important cellular events, more efficient and selective synthetic protocols must be developed for the preparation of oligosaccharides. Classical chemical synthesis is often impractical for the synthesis of complex oligosaccharides because of the need for selective and labor-intensive protection-deprotection steps and difficulties in directing product stereochemistry. To overcome these limitations, enzymatic syntheses using glycosidases or glycosyl transferases have rapidly gained prominence (4 -6).In recent years, the glycosynthase approach developed in this laboratory has added a new dimension to the enzymatic preparation of oligosaccharides (7-9). Glycosynthases are retaining glycosidase mutants in which the catalytic nucleophile has been converted to a non-nucleophilic residue. These mutants catalyze the formation of glycosidic bonds when glycosyl fluorides with anomeric configuration opposite to that of the original substrate, thereby mimicking the glycosyl enzyme intermediate, are employed as substrates. The modified enzyme catalyzes the nucleophilic displacement of the fluoride via attack by a hydroxyl group on an added glycosyl acceptor, generating a new glycosidic bond with the same stereochemistry as the normal substrate. The reactions catalyzed by glycosynthases are highly amenable to industrial syntheses because of the hig...

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Production of recombinant bacterial endoglucanase as a co-product with ethanol during fermentation using derivatives ofEscherichia coli KO11

Wood

Beall²,

Ingram³

1997

Biotechnol. Bioeng.

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Cellulose-binding polypeptides from Cellulomonas fimi: endoglucanase D (CenD), a family A beta-1,4-glucanase

Cited by 61 publications

References 38 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

Directed Evolution of a Glycosynthase from Agrobacterium sp. Increases Its Catalytic Activity Dramatically and Expands Its Substrate Repertoire

Production of recombinant bacterial endoglucanase as a co-product with ethanol during fermentation using derivatives ofEscherichia coli KO11

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