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Spontaneous Cleavages of a Heterologous Protein, the CenA Endoglucanase of Cellulomonas fimi, in Escherichia coli.

ABSTRACT: CenA is an endoglucanase secreted by the Gram-positive cellulolytic bacterium, Cellulomonas fimi, to the environment as a glycosylated protein. The role of glycosylation in CenA is unclear. However, it seems not crucial for functional activity and secretion since the unglycosylated counterpart, recombinant CenA (rCenA), is both bioactive and secretable in Escherichia coli. Using a systematic screening approach, we have demonstrated that rCenA is subjected to spontaneous cleavages (SC) in both the cytoplasm and culture medium of E. coli, under the influence of different environmental factors. The cleavages were found to occur in both the cellulose-binding (CellBD) and catalytic domains, with a notably higher occurring rate detected in the former than the latter. In CellBD, the cleavages were shown to occur close to potential N-linked glycosylation sites, suggesting that these sites might serve as 'attributive tags' for differentiating rCenA from endogenous proteins and the points of initiation of SC. It is hypothesized that glycosylation plays a crucial role in protecting CenA from SC when interacting with cellulose in the environment. Subsequent to hydrolysis, SC would ensure the dissociation of CenA from the enzyme-substrate complex. Thus, our findings may help elucidate the mechanisms of protein turnover and enzymatic cellulolysis.

SUBMITTER: Lai CY

PROVIDER: S-EPMC8209791 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Mannan-degrading enzymes from Cellulomonas fimi.

Project description:The genes man26a and man2A from Cellulomonas fimi encode mannanase 26A (Man26A) and beta-mannosidase 2A (Man2A), respectively. Mature Man26A is a secreted, modular protein of 951 amino acids, comprising a catalytic module in family 26 of glycosyl hydrolases, an S-layer homology module, and two modules of unknown function. Exposure of Man26A produced by Escherichia coli to C. fimi protease generates active fragments of the enzyme that correspond to polypeptides with mannanase activity produced by C. fimi during growth on mannans, indicating that it may be the only mannanase produced by the organism. A significant fraction of the Man26A produced by C. fimi remains cell associated. Man2A is an intracellular enzyme comprising a catalytic module in a subfamily of family 2 of the glycosyl hydrolases that at present contains only mammalian beta-mannosidases.

| S-EPMC91384 | biostudies-literature

Proteomic Analysis of the Secretome of Cellulomonas fimi ATCC 484 and Cellulomonas flavigena ATCC 482.

Project description:The bacteria in the genus Cellulomonas are known for their ability to degrade plant cell wall biomass. Cellulomonas fimi ATCC 484 and C. flavigena ATCC 482 have been the subject of much research into secreted cellulases and hemicellulases. Recently the genome sequences of both C. fimi ATCC 484 and C. flavigena ATCC 482 were published, and a genome comparison has revealed their full spectrum of possible carbohydrate-active enzymes (CAZymes). Using mass spectrometry, we have compared the proteins secreted by C. fimi and C. flavigena during growth on the soluble cellulose substrate, carboxymethylcellulose (CMC), as well as a soluble xylan fraction. Many known C. fimi CAZymes were detected, which validated our analysis, as were a number of new CAZymes and other proteins that, though identified in the genome, have not previously been observed in the secretome of either organism. Our data also shows that many of these are co-expressed on growth of either CMC or xylan. This analysis provides a new perspective on Cellulomonas enzymes and provides many new CAZyme targets for characterization.

| S-EPMC4780727 | biostudies-literature

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

Project description:Five cellulose-binding polypeptides were detected in Cellulomonas fimi culture supernatants. Two of them are CenA and CenB, endo-beta-1,4-glucanases which have been characterized previously; the other three were previously uncharacterized polypeptides with apparent molecular masses of 120, 95, and 75 kDa. The 75-kDa cellulose-binding protein was designated endoglucanase D (CenD). The cenD gene was cloned and sequenced. It encodes a polypeptide of 747 amino acids. Mature CenD is 708 amino acids long and has a predicted molecular mass of 74,982 Da. Analysis of the predicted amino acid sequence of CenD shows that the enzyme comprises four domains which are separated by short linker polypeptides: an N-terminal catalytic domain of 405 amino acids, two repeated sequences of 95 amino acids each, and a C-terminal domain of 105 amino acids which is > 50% identical to the sequences of cellulose-binding domains in Cex, CenA, and CenB from C. fimi. Amino acid sequence comparison placed the catalytic domain of CenD in family A, subtype 1, of beta-1,4-glycanases. The repeated sequences are more than 40% identical to the sequences of three repeats in CenB and are related to the repeats of fibronectin type III. CenD hydrolyzed the beta-1,4-glucosidic bond with retention of anomeric configuration. The activities of CenD towards various cellulosic substrates were quite different from those of CenA and CenB.

| S-EPMC204259 | biostudies-other

Cellulomonas fimi

Project description:Cellulomonas fimi overview

| PRJNA37205 | ENA

Characterization of five β-glycoside hydrolases from Cellulomonas fimi ATCC 484.

Project description:The Gram-positive bacterium Cellulomonas fimi produces a large array of carbohydrate-active enzymes. Analysis of the collection of carbohydrate-active enzymes from the recent genome sequence of C. fimi ATCC 484 shows a large number of uncharacterized genes for glycoside hydrolase (GH) enzymes potentially involved in biomass utilization. To investigate the enzymatic activity of potential β-glucosidases in C. fimi, genes encoding several GH3 enzymes and one GH1 enzyme were cloned and recombinant proteins were expressed in Escherichia coli. Biochemical analysis of these proteins revealed that the enzymes exhibited different substrate specificities for para-nitrophenol-linked substrates (pNP), disaccharides, and oligosaccharides. Celf_2726 encoded a bifunctional enzyme with β-d-xylopyranosidase and α-l-arabinofuranosidase activities, based on pNP-linked substrates (CfXyl3A). Celf_0140 encoded a β-d-glucosidase with activity on β-1,3- and β-1,6-linked glucosyl disaccharides as well as pNP-β-Glc (CfBgl3A). Celf_0468 encoded a β-d-glucosidase with hydrolysis of pNP-β-Glc and hydrolysis/transglycosylation activities only on β-1,6-linked glucosyl disaccharide (CfBgl3B). Celf_3372 encoded a GH3 family member with broad aryl-β-d-glycosidase substrate specificity. Celf_2783 encoded the GH1 family member (CfBgl1), which was found to hydrolyze pNP-β-Glc/Fuc/Gal, as well as cellotetraose and cellopentaose. CfBgl1 also had good activity on β-1,2- and β-1,3-linked disaccharides but had only very weak activity on β-1,4/6-linked glucose.

| S-EPMC4248878 | biostudies-literature

Substrate specificity of endoglucanase A from Cellulomonas fimi: fundamental differences between endoglucanases and exoglucanases from family 6.

Project description:Values of kcat. and Km for the hydrolysis of cellotetraose, cellotriose, beta-cellobiosyl fluoride and various beta-aryl cellobiosides by endoglucanase A (CenA) from Cellulomonas fimi indicate that specific binding interactions between the reducing-end glucose residues of cellotetraose and cellotriose and the enzyme at the transition state provide enormous stabilization, endowing glucose with the "effective leaving group ability' of 2,4-dinitrophenol. As has been seen with several other inverting glycosidases, CenA hydrolyses the "wrong' anomer of its glycosyl fluoride substrate, alpha-cellobiosyl fluoride, according to non-Michaelian kinetics. This indicates that CenA carries out this hydrolysis by a mechanism involving binding of two substrate molecules in the active site (Hehre, Brewer and Genghof (1979) J. Biol. Chem. 254, 5942-5950] in contrast with that reported for cellobiohydrolase II, another family-6 enzyme [Konstantinidis, Marsden and Sinnott (1993) Biochem. J. 291, 833-838]. The pH profiles for wild-type CenA indicate that kcat. for CenA depends on the presence of both a protonated group and a deprotonated group for full activity, consistent with the presence of an acid and a base catalyst at the active site. By contrast, the profile for the Asp252Ala mutant of CenA shows a dependence only on a base-catalytic group, thereby confirming the role of Asp-252 as an acid catalyst. These results show that hydrolysis by CenA occurs by a typical inverting mechanism involving both acid and base catalysis, as first proposed by Koshland. It also suggests that endoglucanases from family 6 may function by fundamentally different mechanisms for exoglucanases in this family.

| S-EPMC1217219 | biostudies-other

Characterisation of novel biomass degradation enzymes from the genome of Cellulomonas fimi.

Project description:Recent analyses of genome sequences belonging to cellulolytic bacteria have revealed many genes potentially coding for cellulosic biomass degradation enzymes. Annotation of these genes however, is based on few biochemically characterised examples. Here we present a simple strategy based on BioBricks for the rapid screening of candidate genes expressed in Escherichia coli. As proof of principle we identified over 70 putative biomass degrading genes from bacterium Cellulomonas fimi, expressing a subset of these in BioBrick format. Six novel genes showed activity in E. coli. Four interesting enzymes were characterised further. α-l-arabinofuranosidase AfsB, β-xylosidases BxyF and BxyH and multi-functional β-cellobiosidase/xylosidase XynF were partially purified to determine their optimum pH, temperature and kinetic parameters. One of these enzymes, BxyH, was unexpectedly found to be highly active at strong alkaline pH and at temperatures as high as 100 °C. This report demonstrates a simple method of quickly screening and characterising putative genes as BioBricks.

| S-EPMC5892457 | biostudies-literature

A self-inducible heterologous protein expression system in Escherichia coli.

Project description:Escherichia coli is an important experimental, medical and industrial cell factory for recombinant protein production. The inducible lac promoter is one of the most commonly used promoters for heterologous protein expression in E. coli. Isopropyl-?-D-thiogalactoside (IPTG) is currently the most efficient molecular inducer for regulating this promoter's transcriptional activity. However, limitations have been observed in large-scale and microplate production, including toxicity, cost and culture monitoring. Here, we report the novel SILEX (Self-InducibLe Expression) system, which is a convenient, cost-effective alternative that does not require cell density monitoring or IPTG induction. We demonstrate the broad utility of the presented self-inducible method for a panel of diverse proteins produced in large amounts. The SILEX system is compatible with all classical culture media and growth temperatures and allows protein expression modulation. Importantly, the SILEX system is proven to be efficient for protein expression screening on a microplate scale.

| S-EPMC5017159 | biostudies-literature

The Product of the fimI gene is necessary for Escherichia coli type 1 pilus biosynthesis.

Project description:Site-directed mutagenesis was employed to create lesions in fimI, a gene of uncertain function located in the chromosomal gene cluster (fim) involved in Escherichia coli type 1 pilus biosynthesis. Chromosomal fimI mutations produced a piliation-negative phenotype. Complementation analysis indicated that a fimI'-kan insertion mutation and a fimI frameshift mutation produced polarity-like effects not seen with an in-frame fimI deletion mutation. Minicell analysis associated fimI with a 16.4-kDa noncytoplasmic protein product (FimI). We conclude that FimI has a required role in normal pilus biosynthesis.

| S-EPMC166486 | biostudies-literature

The genome sequences of Cellulomonas fimi and "Cellvibrio gilvus" reveal the cellulolytic strategies of two facultative anaerobes, transfer of "Cellvibrio gilvus" to the genus Cellulomonas, and proposal of Cellulomonas gilvus sp. nov.

Project description:Actinobacteria in the genus Cellulomonas are the only known and reported cellulolytic facultative anaerobes. To better understand the cellulolytic strategy employed by these bacteria, we sequenced the genome of the Cellulomonas fimi ATCC 484(T). For comparative purposes, we also sequenced the genome of the aerobic cellulolytic "Cellvibrio gilvus" ATCC 13127(T). An initial analysis of these genomes using phylogenetic and whole-genome comparison revealed that "Cellvibrio gilvus" belongs to the genus Cellulomonas. We thus propose to assign "Cellvibrio gilvus" to the genus Cellulomonas. A comparative genomics analysis between these two Cellulomonas genome sequences and the recently completed genome for Cellulomonas flavigena ATCC 482(T) showed that these cellulomonads do not encode cellulosomes but appear to degrade cellulose by secreting multi-domain glycoside hydrolases. Despite the minimal number of carbohydrate-active enzymes encoded by these genomes, as compared to other known cellulolytic organisms, these bacteria were found to be proficient at degrading and utilizing a diverse set of carbohydrates, including crystalline cellulose. Moreover, they also encode for proteins required for the fermentation of hexose and xylose sugars into products such as ethanol. Finally, we found relatively few significant differences between the predicted carbohydrate-active enzymes encoded by these Cellulomonas genomes, in contrast to previous studies reporting differences in physiological approaches for carbohydrate degradation. Our sequencing and analysis of these genomes sheds light onto the mechanism through which these facultative anaerobes degrade cellulose, suggesting that the sequenced cellulomonads use secreted, multidomain enzymes to degrade cellulose in a way that is distinct from known anaerobic cellulolytic strategies.

| S-EPMC3544764 | biostudies-literature

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