Differences in the chitinolytic activity of mammalian chitinases on soluble and crystalline substrates

AbstractChitin is an abundant polysaccharide used by a large range of organisms for structural rigidity and water repulsion. As such, the insoluble crystalline structure of chitin poses significant challenges for enzymatic degradation. Vertebrates do not produce chitin, but do express chitin degrading enzymes. Acidic mammalian chitinase, the primary enzyme involved in the degradation of environmental chitin in mammalian lungs, is a processive glycosyl hydrolase that may be able to make multiple hydrolysis events for each binding event. Mutations to acidic mammalian chitinase have been associated with asthma, and genetic deletion of the enzyme in mice results in significantly increased morbidity and mortality with age. We initially set out to reverse this phenotype by engineering hyperactive acidic mammalian chitinase variants. Using a directed evolution screening approach using commercial fluorogenic substrates, we identified mutations with consistent increases in activity. To determine whether the activity increases observed with oligomeric substrates were consistent with more biologically relevant chitin substrates, we developed new assays to quantify chitinase activity with colloidal crystalline chitin, and identified a high throughput fluorogenic assay that gives sufficient signal to noise advantages to quantify changes to activity due to the addition or removal of a chitin binding domain to the enzyme. We show that the activity increasing mutations derived from our directed evolution screen were lost when crystalline substrates were used. In contrast, naturally occurring gain-of-function mutations gave similar results with oligomeric and crystalline substrates. We also show that the activity differences between acidic mammalian chitinase and chitotriosidase are reduced in the context of crystalline substrate, suggesting that previously reported activity differences with oligomeric substrates may have been largely driven by differential substrate specificity for the oligomers. These results highlight the need for assays against more physiological substrates when engineering complex metabolic enzymes, and provide a new approach that may be broadly applicable to engineering glycosyl hydrolases.

Download Full-text

Growth of Azospirillum irakense KBC1 on the Aryl β-Glucoside Salicin Requires either salA or salB

Journal of Bacteriology ◽

10.1128/jb.181.10.3003-3009.1999 ◽

1999 ◽

Vol 181 (10) ◽

pp. 3003-3009 ◽

Cited By ~ 12

Author(s):

Denis Faure ◽

Jos Desair ◽

Veerle Keijers ◽

My Ali Bekri ◽

Paul Proost ◽

...

Keyword(s):

Microbial Growth ◽

Glycosyl Hydrolase ◽

Cosmid Library ◽

Glycosyl Hydrolases ◽

Amino Acid Similarity ◽

Functional Homology ◽

Low Degree ◽

Genetic Implication ◽

Glycosyl Hydrolase Family 3 ◽

Hydrolase Family

ABSTRACT The rhizosphere nitrogen-fixing bacteriumAzospirillum irakense KBC1 is able to grow on pectin and β-glucosides such as cellobiose, arbutin, and salicin. Two adjacent genes, salA and salB, conferring β-glucosidase activity to Escherichia coli, have been identified in a cosmid library of A. irakense DNA. The SalA and SalB enzymes preferentially hydrolyzed aryl β-glucosides. A Δ(salA-salB) A. irakense mutant was not able to grow on salicin but could still utilize arbutin, cellobiose, and glucose for growth. This mutant could be complemented by either salA or salB, suggesting functional redundancy of these genes in salicin utilization. In contrast to this functional homology, the SalA and SalB proteins, members of family 3 of the glycosyl hydrolases, show a low degree of amino acid similarity. Unlike SalA, the SalB protein exhibits an atypical truncated C-terminal region. We propose that SalA and SalB are representatives of the AB and AB′ subfamilies, respectively, in glycosyl hydrolase family 3. This is the first genetic implication of this β-glucosidase family in the utilization of β-glucosides for microbial growth.

Download Full-text

Molecular Analysis of the Gene Encoding a Novel Chitin-Binding Protease from Alteromonas sp. Strain O-7 and Its Role in the Chitinolytic System

Journal of Bacteriology ◽

10.1128/jb.184.7.1865-1872.2002 ◽

2002 ◽

Vol 184 (7) ◽

pp. 1865-1872 ◽

Cited By ~ 18

Author(s):

Katsushiro Miyamoto ◽

Eiji Nukui ◽

Hiroyuki Itoh ◽

Takaji Sato ◽

Takeshi Kobayashi ◽

...

Keyword(s):

Molecular Mass ◽

Signal Sequence ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Chitinase Activity ◽

Maximum Activity ◽

Sequencing Analysis ◽

Gene Encoding ◽

Calculated Molecular Mass ◽

Chitin Binding Domain

ABSTRACT Alteromonas sp. strain O-7 secretes several proteins in response to chitin induction. We have found that one of these proteins, designated AprIV, is a novel chitin-binding protease involved in chitinolytic activity. The gene encoding AprIV (aprIV) was cloned in Escherichia coli. DNA sequencing analysis revealed that the open reading frame of aprIV encoded a protein of 547 amino acids with a calculated molecular mass of 57,104 Da. AprIV is a modular enzyme consisting of five domains: the signal sequence, the N-terminal proregion, the family A subtilase region, the polycystic kidney disease domain (PkdD), and the chitin-binding domain type 3 (ChtBD3). Expression plasmids coding for PkdD or both PkdD and ChtBD (PkdD-ChtBD) were constructed. The PkdD-ChtBD but not PkdD exhibited strong binding to α-chitin and β-chitin. Western and Northern analyses demonstrated that aprIV was induced in the presence of N-acetylglucosamine, N-acetylchitobiose, or chitin. Native AprIV was purified to homogeneity from Alteromonas sp. strain O-7 and characterized. The molecular mass of mature AprIV was estimated to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature of AprIV were pH 11.5 and 35°C, respectively, and even at 10°C the enzyme showed 25% of the maximum activity. Pretreatment of native chitin with AprIV significantly promoted chitinase activity.

Download Full-text

Marked Differences in Tissue-specific Expression of Chitinases in Mouse and Man

Journal of Histochemistry & Cytochemistry ◽

10.1369/jhc.4a6547.2005 ◽

2005 ◽

Vol 53 (10) ◽

pp. 1283-1292 ◽

Cited By ~ 96

Author(s):

Rolf G. Boot ◽

Anton P. Bussink ◽

Marri Verhoek ◽

Piet A.J. de Boer ◽

Antoon F.M. Moorman ◽

...

Keyword(s):

Gastrointestinal Tract ◽

Species Difference ◽

Chitinase Activity ◽

Specific Enzyme ◽

Specific Expression ◽

Tissue Specific ◽

Tissue Specific Expression ◽

Pathological Conditions ◽

Acidic Mammalian Chitinase ◽

Promoter Usage

Two distinct chitinases have been identified in mammals: a phagocyte-specific enzyme named chitotriosidase and an acidic mammalian chitinase (AMCase) expressed in the lungs and gastrointestinal tract. Increased expression of both chitinases has been observed in different pathological conditions: chitotriosidase in lysosomal lipid storage disorders like Gaucher disease and AMCase in asthmatic lung disease. Recently, it was reported that AMCase activity is involved in the pathogenesis of asthma in an induced mouse model. Inhibition of chitinase activity was found to alleviate the inflammation-driven pathology. We studied the tissue-specific expression of both chitinases in mice and compared it to the situation in man. In both species AMCase is expressed in alveolar macrophages and in the gastrointestinal tract. In mice, chitotriosidase is expressed only in the gastrointestinal tract, the tongue, fore-stomach, and Paneth cells in the small intestine, whereas in man the enzyme is expressed exclusively by professional phagocytes. This species difference seems to be mediated by distinct promoter usage. In conclusion, the pattern of expression of chitinases in the lung differs between mouse and man. The implications for the development of anti-asthma drugs with chitinases as targets are discussed.

Download Full-text

Characterization of an Exo-β-d-Glucosaminidase Involved in a Novel Chitinolytic Pathway from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

Journal of Bacteriology ◽

10.1128/jb.185.17.5175-5181.2003 ◽

2003 ◽

Vol 185 (17) ◽

pp. 5175-5181 ◽

Cited By ~ 69

Author(s):

Takeshi Tanaka ◽

Toshiaki Fukui ◽

Haruyuki Atomi ◽

Tadayuki Imanaka

Keyword(s):

Escherichia Coli ◽

Central Region ◽

Primary Structure ◽

Glycosyl Hydrolase ◽

Chitinase Gene ◽

Glycosyl Hydrolases ◽

Galactosidase Activity ◽

Hyperthermophilic Archaeon ◽

Candidate Protein

ABSTRACT We previously clarified that the chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 produces diacetylchitobiose (GlcNAc2) as an end product from chitin. Here we sought to identify enzymes in T. kodakaraensis that were involved in the further degradation of GlcNAc2. Through a search of the T. kodakaraensis genome, one candidate gene identified as a putative β-glycosyl hydrolase was found in the near vicinity of the chitinase gene. The primary structure of the candidate protein was homologous to the β-galactosidases in family 35 of glycosyl hydrolases at the N-terminal region, whereas the central region was homologous to β-galactosidases in family 42. The purified protein from recombinant Escherichia coli clearly showed an exo-β-d-glucosaminidase (GlcNase) activity but not β-galactosidase activity. This GlcNase (GlmA Tk ), a homodimer of 90-kDa subunits, exhibited highest activity toward reduced chitobiose at pH 6.0 and 80°C and specifically cleaved the nonreducing terminal glycosidic bond of chitooligosaccharides. The GlcNase activity was also detected in T. kodakaraensis cells, and the expression of GlmA Tk was induced by GlcNAc2 and chitin, strongly suggesting that GlmA Tk is involved in chitin catabolism in T. kodakaraensis. These results suggest that T. kodakaraensis, unlike other organisms, possesses a novel chitinolytic pathway where GlcNAc2 from chitin is first deacetylated and successively hydrolyzed to glucosamine. This is the first report that reveals the primary structure of GlcNase not only from an archaeon but also from any organism.

Download Full-text

Plant vascular system-feeding Psyllidae (Hemiptera) and Nematoda genomes encode family 12 glycosyl hydrolases

The Canadian Entomologist ◽

10.4039/tce.2019.11 ◽

2019 ◽

Vol 151 (3) ◽

pp. 291-297

Author(s):

Richard W. Jones

Keyword(s):

Plant Cell Wall ◽

Vascular Tissue ◽

Vascular System ◽

Glycosyl Hydrolase ◽

Expression System ◽

Root Tip ◽

Glycosyl Hydrolases ◽

Endoglucanase Activity ◽

Intron Structure ◽

Hydrolase Family

AbstractInsect-encoded cellulolytic plant cell wall hydrolases have thus far been found mostly from glycosyl hydrolase family 5, 9, 10, and 45. We now report the first evidence for genomic encoding of family 12 glycosyl hydrolases in vascular feeding Psyllidae (Hemiptera) and Nematoda. The genes were identified in three psyllids (Acanthocasuarina muellerianae Taylor, Pachypsylla venusta (Osten-Sacken), and Diaphorina citri Kuwayama) and a root tip feeding dagger nematode (Xiphinema index Thorne and Allen; Dorylaimida: Longidoridae). While the final gene products were highly similar, the genomic intron structure varied, having a 2 kB intron in P. venusta, a 283 base-pair intron in D. citri, and no intron in X. index. Endoglucanase activity was demonstrated using the D. citri genes in an Agrobacterium Conn (Rhizobiaceae) infiltration-based plant expression system. The presence of family 12 endoglucanases in this set of insects suggests a specific role in facilitating feeding on vascular tissue.

Download Full-text

Molecular characterization of a male-specific glycosyl hydrolase, Lma-p72, secreted on to the abdominal surface of the Madeira cockroach Leucophaea maderae (Blaberidae, Oxyhaloinae)

Biochemical Journal ◽

10.1042/bj20030025 ◽

2003 ◽

Vol 372 (2) ◽

pp. 535-541 ◽

Cited By ~ 22

Author(s):

Richard CORNETTE ◽

Jean-Pierre FARINE ◽

Dehbia ABED-VIELLARD ◽

Brigitte QUENNEDEY ◽

Rémy BROSSUT

Keyword(s):

Glycosyl Hydrolase ◽

Surface Protein ◽

Glycosyl Hydrolases ◽

Adult Males ◽

Reverse Transcription Pcr ◽

Sternal Glands ◽

Leucophaea Maderae ◽

Abdominal Surface ◽

Male Specific

The epicuticular surface protein Lma-p72 is specific to the abdominal secretions of Leucophaea maderae (Madeira cockroach) adult males. Natural Lma-p72 was purified and the complete cDNA sequence determined by reverse-transcription PCR using primers based on Edman degradation fragments. Northern blot and in situ hybridization analyses showed that Lma-p72 was expressed in the tergal and sternal glands. Sequence alignment indicates that Lma-p72 is closely related to the family 1 glycosyl hydrolases (EC 3.2.1). Native Lma-p72 was proved to be active in the abdominal secretions and exhibit a β-galactosidase-like activity. However, weak specificity with respect to the C-4 configuration of the substrate was observed. Two main hypotheses were proposed concerning the function of this enzyme: Lma-p72 could hydrolyse oligosaccharides from the male abdominal secretions, making them more phagostimulatory for the female during the precopulatory behaviour. The protein could also cleave a pheromone–sugar conjugate to release the pheromonal compounds on to the cuticular surface. Such a sugar conjugate could be a transport form. Data from the first in vivo inhibition tests indicate that a glycosidase could be directly involved in the production process of some pheromonal compounds in L. maderae males.

Download Full-text

Chitinase Induction Prior to Caspofungin Treatment of Experimental Invasive Aspergillosis in Neutropenic Rats Does Not Enhance Survival

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00960-17 ◽

2017 ◽

Vol 62 (1) ◽

Author(s):

Jeannine M. Refos ◽

Alieke G. Vonk ◽

Marian T. ten Kate ◽

Henri A. Verbrugh ◽

Irma A. J. M. Bakker-Woudenberg ◽

...

Keyword(s):

Antifungal Activity ◽

Invasive Aspergillosis ◽

Invasive Pulmonary Aspergillosis ◽

Left Lung ◽

Chitinase Activity ◽

Pulmonary Aspergillosis ◽

Content Type ◽

Acidic Mammalian Chitinase ◽

Phosphate Buffered Saline

ABSTRACT Host chitinases, chitotriosidase and acidic mammalian chitinase (AMCase), improved the antifungal activity of caspofungin (CAS) against Aspergillus fumigatus in vitro. These chitinases are not constitutively expressed in the lung. Here, we investigated whether chitosan derivatives were able to induce chitinase activity in the lungs of neutropenic rats and, if so, whether these chitinases were able to prolong survival of rats with invasive pulmonary aspergillosis (IPA) or of rats with IPA and treated with CAS. An oligosaccharide-lactate chitosan (OLC) derivative was instilled in the left lung of neutropenic rats to induce chitotriosidase and AMCase activities. Rats instilled with OLC or with phosphate-buffered saline (PBS) were subsequently infected with A. fumigatus and then treated with suboptimal doses of CAS. Survival, histopathology, and galactomannan indexes were determined. Instillation of OLC resulted in chitotriosidase and AMCase activities. However, instillation of OLC did not prolong rat survival when rats were subsequently challenged with A. fumigatus. In 5 of 7 rats instilled with OLC, the fungal foci in the lungs were smaller than those in rats instilled with PBS. Instillation of OLC did not significantly enhance the survival of neutropenic rats challenged with A. fumigatus and treated with a suboptimal dosage of CAS. Chitotriosidase and AMCase activities can be induced with OLC, but the presence of active chitinases in the lung did not prevent the development of IPA or significantly enhance the therapeutic outcome of CAS treatment.

Download Full-text

Characterization of a highly xylose tolerant β-xylosidase isolated from high temperature horse manure compost

BMC Biotechnology ◽

10.1186/s12896-021-00722-6 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Kanyisa Ndata ◽

Walter Nevondo ◽

Bongi Cekuse ◽

Leonardo Joaquim van Zyl ◽

Marla Trindade

Keyword(s):

Glycosyl Hydrolase ◽

Metagenomic Library ◽

Bioinformatic Analysis ◽

Glycoside Hydrolase Family ◽

Kinetic Properties ◽

Glycosyl Hydrolases ◽

Remarkable Feature ◽

Manure Compost ◽

Horse Manure ◽

Hydrolase Family

Abstract Background There is a continued need for improved enzymes for industry. β-xylosidases are enzymes employed in a variety of industries and although many wild-type and engineered variants have been described, enzymes that are highly tolerant of the products produced by catalysis are not readily available and the fundamental mechanisms of tolerance are not well understood. Results Screening of a metagenomic library constructed of mDNA isolated from horse manure compost for β-xylosidase activity identified 26 positive hits. The fosmid clones were sequenced and bioinformatic analysis performed to identity putative β-xylosidases. Based on the novelty of its amino acid sequence and potential thermostability one enzyme (XylP81) was selected for expression and further characterization. XylP81 belongs to the family 39 β-xylosidases, a comparatively rarely found and characterized GH family. The enzyme displayed biochemical characteristics (KM—5.3 mM; Vmax—122 U/mg; kcat—107; Topt—50 °C; pHopt—6) comparable to previously characterized glycoside hydrolase family 39 (GH39) β-xylosidases and despite nucleotide identity to thermophilic species, the enzyme displayed only moderate thermostability with a half-life of 32 min at 60 °C. Apart from acting on substrates predicted for β-xylosidase (xylobiose and 4-nitrophenyl-β-D-xylopyranoside) the enzyme also displayed measurable α-L-arabainofuranosidase, β-galactosidase and β-glucosidase activity. A remarkable feature of this enzyme is its ability to tolerate high concentrations of xylose with a Ki of 1.33 M, a feature that is highly desirable for commercial applications. Conclusions Here we describe a novel β-xylosidase from a poorly studied glycosyl hydrolase family (GH39) which despite having overall kinetic properties similar to other bacterial GH39 β-xylosidases, displays unusually high product tolerance. This trait is shared with only one other member of the GH39 family, the recently described β-xylosidases from Dictyoglomus thermophilum. This feature should allow its use as starting material for engineering of an enzyme that may prove useful to industry and should assist in the fundamental understanding of the mechanism by which glycosyl hydrolases evolve product tolerance.

Download Full-text

Classification of glycosyl hydrolases based on structural homology

Journal of Universal Science Online ◽

10.17202/juso.2015.2.1 ◽

2015 ◽

Vol 2 (1) ◽

pp. 1-9

Author(s):

László Fülöp ◽

Tamás Ponyi

Keyword(s):

Amino Acid ◽

Classification System ◽

Catalytic Domain ◽

Graphical Representation ◽

Glycosyl Hydrolase ◽

Evolutionary Relationship ◽

Structural Similarity ◽

Glycosyl Hydrolases ◽

Automated Method

Glycosyl hydrolases are a well-known group of enzymes, which hydrolyze the glycosidic bond between carbohydrates, or between a carbohydrate and different molecules. Glycosyl hydrolases play a vital role in the human body, and are widely used in industrial applications. Glycosyl hydrolases classification is based on substrate specificity and amino acid or nucleotide sequence similarity which reflects their evolutionary relationship. Our aim, in this study, was to carry out the classification of glycosyl hydrolases, based solely on structural similarity which was made possible by the several structures available in the databases and the availability of computing power to conduct such a computationally intensive task, in a reasonable time-frame. It was also aimed that the structural similarity based classification be compared to the present classification system. Based on an all-against-all comparison, we conducted a structural comparison of glycosyl hydrolases. The results are presented graphically. The graphical representation defined 24 structurally homologous classes. The classification was validated using Cα - Cα distance analysis and amino acid sequence cluster analysis. Advantages of this method are that – being an automated method – it is fast, simple and reproducible. Glycosyl hydrolases could be classified into 24 separate classes. N-glycosyl and O-glycosyl hydrolases (both forming binding and catalytic domain classes as well) were clearly different, the former consisting of 8 classes, and the latter consisting of 16 classes. Structural classes simplified the previous classification system. This classification represents the current glycosyl hydrolase family system, but also extends it especially concerning the clan system.

Download Full-text

The chitinolytic activity of the Curtobacterium sp. isolated from field-grown soybean and analysis of its genome sequence

PLoS ONE ◽

10.1371/journal.pone.0259465 ◽

2021 ◽

Vol 16 (11) ◽

pp. e0259465

Author(s):

Ivica Dimkić ◽

Vibha Bhardwaj ◽

Valeria Carpentieri-Pipolo ◽

Nemanja Kuzmanović ◽

Giuliano Degrassi

Keyword(s):

Specific Activity ◽

Gene Clusters ◽

Defense Responses ◽

Chitinase Activity ◽

Glycosyl Hydrolases ◽

Phylogenetic Cluster ◽

Genome Visualization ◽

Chitinase Production ◽

Or Gene ◽

Hydrolysis Of

Curtobacterium sp. GD1 was isolated from leaves of conventionally grown soybean in Brazil. It was noteworthy that among all bacteria previously isolated from the same origin, only Curtobacterium sp. GD1 showed a strong chitinase activity. The enzyme was secreted and its production was induced by the presence of colloidal chitin in the medium. The chitinase was partially purified and characterized: molecular weight was approximately 37 kDa and specific activity 90.8 U/mg. Furthermore, Curtobacterium sp. GD1 genome was sequenced and analyzed. Our isolate formed a phylogenetic cluster with four other Curtobacterium spp. strains, with ANIb/ANIm ≥ 98%, representing a new, still non described Curtobacterium species. The circular genome visualization and comparison of genome sequences of strains forming new cluster indicated that most regions within their genomes were highly conserved. The gene associated with chitinase production was identified and the distribution pattern of glycosyl hydrolases genes was assessed. Also, genes associated with catabolism of structural carbohydrates such as oligosaccharides, mixed polysaccharides, plant and animal polysaccharides, as well as genes or gene clusters associated with resistance to antibiotics, toxic compounds and auxin biosynthesis subsystem products were identified. The abundance of putative glycosyl hydrolases in the genome of Curtobacterium sp. GD1 suggests that it has the tools for the hydrolysis of different polysaccharides. Therefore, Curtobacterium sp. GD1 isolated from soybean might be a bioremediator, biocontrol agent, an elicitor of the plant defense responses or simply degrader.

Download Full-text