The role of carboxyl groups in the function of endo‐β‐1, 4‐glucanase from <i>Schizophyllum commune</i>

Clarke, Anthony J.; Yaguchi, Makoto

doi:10.1111/j.1432-1033.1985.tb08917.x

Cited by 53 publications

(17 citation statements)

References 48 publications

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“…Limited sequence homology to the active site of lysozyme has been found for endo-(CenA) and exo-(Cex) glucanases from C. fimi (3) and a number of fungi (43,55). These findings are consistent with the concept that cellulases act like lysozyme by an acid catalysis mechanism (10).…”

Section: Resultssupporting

confidence: 80%

Structure of the cel-3 gene from Fibrobacter succinogenes S85 and characteristics of the encoded gene product, endoglucanase 3

et al. 1989

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The cel-3 gene cloned from Fibrobacter succinogenes into Escherichia coli coded for the enzyme EG3, which exhibited both endoglucanase and cellobiosidase activities. The gene had an open reading frame of 1,974 base pairs, coding for a protein of 73.4 kilodaltons (kDa). However, the enzyme purified from the osmotic shock fluid of E. coli was 43 kDa. The amino terminus of the 43-kDa protein matched amino acid residue 266 of the protein coded for by the open reading frame, indicating proteolysis in E. coli. In addition to the 43-kDa protein, Western immunoblotting revealed a 94-kDa membranous form of the enzyme in E. coli and a single protein of 118 kDa in F. succinogenes. Thus, the purified protein appears to be a proteolytic degradation product of a native protein which was 94 kDa in E. coli and 118 kDa in F. succinogenes. The discrepancy between the molecular weight expected on the basis of the DNA sequence and the in vivo form may be due to anomalous migration during electrophoresis, to glycosylation of the native enzyme, or to fatty acyl substitution at the N terminus. One of two putative signal peptide cleavage sites bore a strong resemblance to known lipoprotein leader sequences. The purified 43-kDa peptide exhibited a high Km (53 mg/ml) for carboxymethyl cellulose but a low Km (3 to 4 mg/ml) for lichenan and barley I8-glucan. The enzyme hydrolyzed amorphous cellulose, and celiobiose and celiotriose were the major products of hydrolysis. Cellotriose, but not cellobiose, was cleaved by the enzyme. EG3 exhibited signfficant amino acid sequence homology with endoglucanase CelC from Clostridium thermocelum, and as with both CelA and CelC of C. thermocelum, it had a putative active site which could be aligned with the active site of hen egg white lysozyme at the highly conserved amino acid residues Asn-44 and Asp-52.

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Section: Resultssupporting

confidence: 80%

Structure of the cel-3 gene from Fibrobacter succinogenes S85 and characteristics of the encoded gene product, endoglucanase 3

et al. 1989

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“…This reagent readily forms the N-acylurea derivative of protein carboxyl groups (56) since, unlike EDC, it cannot undergo the stabilizing tautomerization to unreactive cyclic forms (54). Compared with other water-soluble carbodiimides, EAC has been utilized relatively little, previous studies comprising the characterization of cytochrome c oxidase (38), enolase (16), cellulase (13) and thrombin (12). The present results underline the advantages in employing EAC for labelling of carboxylic acid residues.…”

Section: Discussionmentioning

confidence: 82%

Chemical modification of carboxyl groups in glucoamylase from Aspergillus niger

Svensson

Møller

Clarke

1988

Carlsberg Res. Commun.

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Glucoamylases GI and G2 from Aspergillus niger lost the catalytic activity by prolonged treatment with 1 -ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC). A tolal of 23 and 16 carboxyl groups were substituted out of 71 and 54 present in the GI and G2 enzyme, respectively. The kinetics and the pH-dependence of the inactivation were compatible with the existence of one essential carboxyl group with a pK~ 5.5. The inhibitor acarbose (a pseudotetrasaccharide) and the substrate maltose prevented EAC-modification of 4 and 2 carboxyl groups, respectively, with considerable retention of enzyme activity. Following removal of these fig,ands, differenlial labelling of critical carboxyl residue(s) was achieved by means of [3H]EAC.Glucoamylase G2 with 12 EAC groups incorporated, prepared in the presence of acarbose, displayed a two-fold increase of K~ and a slight reduction of V,~ for hydrolysis of starch relative to the unmodified enzyme. Both K~ and Vm~ for hydrolysis of maltose were almost unaffected. In addition, maltose and three inhibitors perturbed the UV absorbance spectrum of this derivative. An altered shape of the acarbose-induced difference spectrum implied substitution of a carboxyl group near a binding tryptophanyl residue at a distance from the catalytic site. Efficient protection by acarbose against only the second part of the biphasic course of inactivation similarly reflected that residues playing different roles in the mechanism of action reacted with EAC.In separate experiments, evidence was obtained for a preferred reaction of ethylenimine with enzymatically important carboxyl groups in glucoamylase.

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“…By chemical modification studies, evidence was provided for the importance of carboxyl groups for catalysis in the active site of the Schizophyllum commune xylanase (7). The importance of Glu residues had been suggested by chemical modification of various cellulases (2), including endo-3-1,4-glucanase from S. commune (10), and cellobiohydrolase I from Trichoderma reesei (42). By site-directed mutagenesis, and Glu-169 were changed to the isoteric glutamine form in endo-,B-1,4-glucanase from Bacillus polymyxa and Bacillus subtilis, respectively, resulting in a dramatic loss of carboxymethyl cellulase activity which could be restored by reverse mutation (1).…”

Section: Discussionmentioning

confidence: 99%

Characterization of the active site and thermostability regions of endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI

et al. 1993

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Deletion mutants were constructed from pZEP12, which contained the intact Thermoanaerobacterium saccharolyticum endoxylanase gene (xynA). Deletion of 1.75 kb from the N-terminal end of xynA resulted in a mutant enzyme that retained activity but lost thermostability. Deletion of 1.05 kb from the C terminus did not alter thermostability or activity. The deduced amino acid sequence of T. saccharolyticum B6A-RI endoxylanase XynA was aligned with five other family F beta-glycanases by using the PILEUP program of the Genetics Computer Group package. This multiple alignment of amino acid sequences revealed six highly conserved motifs which included the consensus sequence consisting of a hydrophobic amino acid, Ser or Thr, Glu, a hydrophobic amino acid, Asp, and a hydrophobic amino acid in the catalytic domain. Endoxylanase was inhibited by EDAC [1-(3-dimethylamino propenyl)-3-ethylcarbodiimide hydrochloride], suggesting that Asp and/or Glu was involved in catalysis. Three aspartic acids, two glutamic acids, and one histidine were conserved in all six enzymes aligned. Hydrophobic cluster analysis revealed that two Asp and one Glu occur in the same hydrophobic clusters in T. saccharolyticum B6A-RI endoxylanase and two other enzymes belonging to family F beta-glycanases and suggests their involvement in a catalytic triad. These two Asp and one Glu in XynA from T. saccharolyticum were targeted for analysis by site-specific mutagenesis. Substitution of Asp-537 and Asp-602 by Asn and Glu-600 by Gln completely destroyed endoxylanase activity. These results suggest that these three amino acids form a catalytic triad that functions in a general acid catalysis mechanism.

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The role of carboxyl groups in the function of endo‐β‐1, 4‐glucanase from Schizophyllum commune

Cited by 53 publications

References 48 publications

Structure of the cel-3 gene from Fibrobacter succinogenes S85 and characteristics of the encoded gene product, endoglucanase 3

Structure of the cel-3 gene from Fibrobacter succinogenes S85 and characteristics of the encoded gene product, endoglucanase 3

Chemical modification of carboxyl groups in glucoamylase from Aspergillus niger

Characterization of the active site and thermostability regions of endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI

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