scholarly journals Characterization of a galactosyl-binding protein module from a Cellvibrio japonicus endo-xyloglucanase defines a new family of Carbohydrate Binding Modules

2020 ◽  
pp. AEM.02634-20
Author(s):  
Mohamed A. Attia ◽  
Harry Brumer
Keyword(s):  
Binding Protein ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Substrate Affinity ◽  
Carbohydrate Binding ◽  
Carbohydrate Active Enzymes ◽  
Plant Polysaccharides ◽  
New Family ◽  
Carbohydrate Binding Modules ◽  
Cellvibrio Japonicus

Carbohydrate-binding modules (CBMs) are usually appended to carbohydrate-active enzymes (CAZymes) and serve to potentiate catalytic activity, e.g. by increasing substrate affinity. The Gram-negative soil saprophyte Cellvibrio japonicus is valuable source for CAZyme and CBM discovery and characterization, due to its innate ability to degrade a wide array of plant polysaccharides. Bioinformatic analysis of the CJA_2959 gene product from C. japonicus revealed a modular architecture consisting of a fibronectin type III (Fn3) module, a cryptic module of unknown function (“X181”), and a Glycoside Hydrolase Family 5 subfamily 4 (GH5_4) catalytic module. We previously demonstrated that the last of these, CjGH5F, is an efficient and specific endo-xyloglucanase [Attia et al. 2018. Biotechnol. Biofuels, 11: 45]. In the present study, C-terminal fusion of superfolder green fluorescent protein in tandem with the Fn3-X181 modules enabled recombinant production and purification from Escherichia coli. Native affinity gel electrophoresis revealed binding specificity for the terminal galactose-containing plant polysaccharides galactoxyloglucan and galactomannan. Isothermal titration calorimetry further evidenced a preference for galactoxyloglucan polysaccharide over short oligosaccharides comprising the limit-digest product of CjGH5F. Thus, our results identify the X181 module as the defining member of a new CBM family, CBM88. In addition to directly revealing the function of this CBM in the context of xyloglucan metabolism by C. japonicus, this study will guide future bioinformatic and functional analyses across microbial (meta)genomes.Importance This study reveals Carbohydrate Binding Module Family 88 (CBM88) as a new family of galactose-binding protein modules, which are found in series with diverse microbial glycoside hydrolases, polysaccharide lyases, and carbohydrate esterases. The definition of CBM88 in the Carbohydrate-Active Enzymes classification (http://www.cazy.org/CBM88.html) will significantly enable future microbial (meta)genome analysis and functional studies.

scholarly journals The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation

2003 ◽  
Vol 371 (3) ◽  
pp. 1027-1043 ◽  
Author(s):  
Deborah HOGG ◽  
Gavin PELL ◽  
Paul DUPREE ◽  
Florence GOUBET ◽  
Susana M. MARTÍN-ORÚE ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Glycoside Hydrolases ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Molecular Architecture ◽  
Catalytic Domains ◽  
Carbohydrate Binding Modules ◽  
Cellvibrio Japonicus

β-1,4-Mannanases (mannanases), which hydrolyse mannans and glucomannans, are located in glycoside hydrolase families (GHs) 5 and 26. To investigate whether there are fundamental differences in the molecular architecture and biochemical properties of GH5 and GH26 mannanases, four genes encoding these enzymes were isolated from Cellvibrio japonicus and the encoded glycoside hydrolases were characterized. The four genes, man5A, man5B, man5C and man26B, encode the mannanases Man5A, Man5B, Man5C and Man26B, respectively. Man26B consists of an N-terminal signal peptide linked via an extended serine-rich region to a GH26 catalytic domain. Man5A, Man5B and Man5C contain GH5 catalytic domains and non-catalytic carbohydrate-binding modules (CBMs) belonging to families 2a, 5 and 10; Man5C in addition contains a module defined as X4 of unknown function. The family 10 and 2a CBMs bound to crystalline cellulose and ivory nut crystalline mannan, displaying very similar properties to the corresponding family 10 and 2a CBMs from Cellvibrio cellulases and xylanases. CBM5 bound weakly to these crystalline polysaccharides. The catalytic domains of Man5A, Man5B and Man26B hydrolysed galactomannan and glucomannan, but displayed no activity against crystalline mannan or cellulosic substrates. Although Man5C was less active against glucomannan and galactomannan than the other mannanases, it did attack crystalline ivory nut mannan. All the enzymes exhibited classic endo-activity producing a mixture of oligosaccharides during the initial phase of the reaction, although their mode of action against manno-oligosaccharides and glucomannan indicated differences in the topology of the respective substrate-binding sites. This report points to a different role for GH5 and GH26 mannanases from C. japonicus. We propose that as the GH5 enzymes contain CBMs that bind crystalline polysaccharides, these enzymes are likely to target mannans that are integral to the plant cell wall, while GH26 mannanases, which lack CBMs and rapidly release mannose from polysaccharides and oligosaccharides, target the storage polysaccharide galactomannan and manno-oligosaccharides.

scholarly journals Comparative Analysis of Carbohydrate Active Enzymes in the Flammulina velutipes var. lupinicola Genome

2020 ◽  
Vol 9 (1) ◽  
pp. 20
Author(s):  
Hye-Won Yu ◽  
Ji-Hoon Im ◽  
Won-Sik Kong ◽  
Young-Jin Park
Keyword(s):  
De Novo ◽  
Animal Feed ◽  
Gc Content ◽  
Industrial Applications ◽  
Glycoside Hydrolases ◽  
Flammulina Velutipes ◽  
Carbohydrate Binding ◽  
Carbohydrate Active Enzymes ◽  
Carbohydrate Binding Modules ◽  
Genes Encoding

The purpose of this study was to determine the genome sequence of Flammulina velutipes var. lupinicola based on next-generation sequencing (NGS) and to identify the genes encoding carbohydrate-active enzymes (CAZymes) in the genome. The optimal assembly (71 kmer) based on ABySS de novo assembly revealed a total length of 33,223,357 bp (49.53% GC content). A total of 15,337 gene structures were identified in the F.velutipes var. lupinicola genome using ab initio gene prediction method with Funannotate pipeline. Analysis of the orthologs revealed that 11,966 (96.6%) out of the 15,337 predicted genes belonged to the orthogroups and 170 genes were specific for F. velutipes var. lupinicola. CAZymes are divided into six classes: auxiliary activities (AAs), glycosyltransferases (GTs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), glycoside hydrolases (GHs), and carbohydrate-binding modules (CBMs). A total of 551 genes encoding CAZymes were identified in the F. velutipes var. lupinicola genome by analyzing the dbCAN meta server database (HMMER, Hotpep, and DIAMOND searches), which consisted of 54–95 AAs, 145–188 GHs, 55–73 GTs, 6–19 PLs, 13–59 CEs, and 7–67 CBMs. CAZymes can be widely used to produce bio-based products (food, paper, textiles, animal feed, and biofuels). Therefore, information about the CAZyme repertoire of the F. velutipes var. lupinicola genome will help in understanding the lignocellulosic machinery and in-depth studies will provide opportunities for using this fungus for biotechnological and industrial applications.

scholarly journals Insights into Plant Cell Wall Degradation from the Genome Sequence of the Soil Bacterium Cellvibrio japonicus

2008 ◽  
Vol 190 (15) ◽  
pp. 5455-5463 ◽  
Author(s):  
Robert T. DeBoy ◽  
Emmanuel F. Mongodin ◽  
Derrick E. Fouts ◽  
Louise E. Tailford ◽  
Hoda Khouri ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Hydrolytic Enzymes ◽  
Soil Bacterium ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules ◽  
Cellvibrio Japonicus ◽  
And Storage

ABSTRACT The plant cell wall, which consists of a highly complex array of interconnecting polysaccharides, is the most abundant source of organic carbon in the biosphere. Microorganisms that degrade the plant cell wall synthesize an extensive portfolio of hydrolytic enzymes that display highly complex molecular architectures. To unravel the intricate repertoire of plant cell wall-degrading enzymes synthesized by the saprophytic soil bacterium Cellvibrio japonicus, we sequenced and analyzed its genome, which predicts that the bacterium contains the complete repertoire of enzymes required to degrade plant cell wall and storage polysaccharides. Approximately one-third of these putative proteins (57) are predicted to contain carbohydrate binding modules derived from 13 of the 49 known families. Sequence analysis reveals approximately 130 predicted glycoside hydrolases that target the major structural and storage plant polysaccharides. In common with that of the colonic prokaryote Bacteroides thetaiotaomicron, the genome of C. japonicus is predicted to encode a large number of GH43 enzymes, suggesting that the extensive arabinose decorations appended to pectins and xylans may represent a major nutrient source, not just for intestinal bacteria but also for microorganisms that occupy terrestrial ecosystems. The results presented here predict that C. japonicus possesses an extensive range of glycoside hydrolases, lyases, and esterases. Most importantly, the genome of C. japonicus is remarkably similar to that of the gram-negative marine bacterium, Saccharophagus degradans 2-40T. Approximately 50% of the predicted C. japonicus plant-degradative apparatus appears to be shared with S. degradans, consistent with the utilization of plant-derived complex carbohydrates as a major substrate by both organisms.

scholarly journals Alpha-Agarases Define a New Family of Glycoside Hydrolases, Distinct from Beta-Agarase Families

2007 ◽  
Vol 73 (14) ◽  
pp. 4691-4694 ◽  
Author(s):  
Didier Flament ◽  
Tristan Barbeyron ◽  
Murielle Jam ◽  
Philippe Potin ◽  
Mirjam Czjzek ◽  
...  
Keyword(s):  
Gene Product ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Gene Encoding ◽  
New Family ◽  
Carbohydrate Binding Modules ◽  
Type 3

ABSTRACT The gene encoding the α-agarase from “Alteromonas agarilytica” (proposed name) has been cloned and sequenced. The gene product (154 kDa) is unrelated to β-agarases and instead belongs to a new family of glycoside hydrolases (GH96). The α-agarase also displays a complex modularity, with the presence of five thrombospondin type 3 repeats and three carbohydrate-binding modules.

scholarly journals Characterization of a Broad-Specificity β-Glucanase Acting on β-(1,3)-, β-(1,4)-, and β-(1,6)-Glucans That Defines a New Glycoside Hydrolase Family

2012 ◽  
Vol 78 (24) ◽  
pp. 8540-8546 ◽  
Author(s):  
Mickael Lafond ◽  
David Navarro ◽  
Mireille Haon ◽  
Marie Couturier ◽  
Jean-Guy Berrin
Keyword(s):  
Podospora Anserina ◽  
Full Length ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
High Sequence Identity ◽  
Content Type ◽  
New Family ◽  
Domain Of Unknown Function ◽  
Type Mode

ABSTRACTHere we report the cloning of thePa_3_10940gene from the coprophilic fungusPodospora anserina, which encodes a C-terminal family 1 carbohydrate binding module (CBM1) linked to a domain of unknown function. The function of the gene was investigated by expression of the full-length protein and a truncated derivative without the CBM1 domain in the yeastPichia pastoris. Using a library of polysaccharides of different origins, we demonstrated that the full-length enzyme displays activity toward a broad range of β-glucan polysaccharides, including laminarin, curdlan, pachyman, lichenan, pustulan, and cellulosic derivatives. Analysis of the products released from polysaccharides revealed that this β-glucanase is an exo-acting enzyme on β-(1,3)- and β-(1,6)-linked glucan substrates and an endo-acting enzyme on β-(1,4)-linked glucan substrates. Hydrolysis of short β-(1,3), β-(1,4), and β-(1,3)/β-(1,4) gluco-oligosaccharides confirmed this striking feature and revealed that the enzyme performs in an exo-type mode on the nonreducing end of gluco-oligosaccharides. Excision of the CBM1 domain resulted in an inactive enzyme on all substrates tested. To our knowledge, this is the first report of an enzyme that displays bifunctional exo-β-(1,3)/(1,6) and endo-β-(1,4) activities toward beta-glucans and therefore cannot readily be assigned to existing Enzyme Commission groups. The amino acid sequence has high sequence identity to hypothetical proteins within the fungal taxa and thus defines a new family of glycoside hydrolases, the GH131 family.

scholarly journals Draft Genomes and Comparative Analysis of Seven Mangrove Rhizosphere-Associated Fungi Isolated From Kandelia obovata and Acanthus ilicifolius

2021 ◽  
Vol 2 ◽  
Author(s):  
Chengcheng Shi ◽  
Jianwei Chen ◽  
Qijin Ge ◽  
Jiahui Sun ◽  
Wenjie Guo ◽  
...  
Keyword(s):  
Gene Clusters ◽  
Glycoside Hydrolases ◽  
Gene Copy ◽  
Carbohydrate Binding ◽  
Type I ◽  
Kandelia Obovata ◽  
Carbohydrate Active Enzymes ◽  
Mangrove Fungi ◽  
Acanthus Ilicifolius ◽  
Carbohydrate Binding Modules

Mangroves are one of the most productive and biologically diverse ecosystems, with unique plants, animals, and microorganisms adapted to the harsh coastal environments. Although fungi are widely distributed in the mangrove ecosystem and they are playing an important role in the decomposition of organic matter, their genomic profiles are still poorly understood. In this study, we isolated seven Ascomycota fungi (Westerdykella dispersa F012, Trichoderma lixii F014, Aspergillus tubingensis F023, Penicillium brefeldianum F032, Neoroussoella solani F033, Talaromyces fuscoviridis F034, and Arthrinium marii F035) from rhizospheres of two mangroves of Kandelia obovata and Acanthus ilicifolius. We sequenced and assembled the whole genome of these fungi, resulting in size ranging from 29 to 48 Mb, while contig N50 from 112 to 833 Kb. We generated six novel fungi genomes except A. tubingensis, and the gene completeness and genome completeness of all seven genomes are higher than 94%. Comparing with non-mangrove fungi, we found Carbohydrate-Binding Modules (CBM32), a subfamily of carbohydrate active enzymes, only detected in two mangrove fungi. Another two subfamilies, Glycoside Hydrolases (GH6) and Polysaccharide Lyases (PL4), were significantly different in gene copy number between K. obovata and A. ilicifolius rhizospheres (P-value 0.041 for GH6, 0.047 for PL4). These findings may indicate an important influence of mangrove environments or hosts on the ability of decomposition in rhizosphere fungi. Secondary metabolite biosynthesis gene clusters were detected and we found the mangrove fungi averagely contain 18 Type I Polyketide (t1pks) synthase, which was significantly higher than 13 in non-mangrove fungi (P-value 0.048), suggesting their potential roles in producing bioactive compounds that important for fungi development and ecology. We reported seven mangrove-associated fungal genomes in this study and compared their carbohydrate active enzymes and secondary metabolites (SM) genes with those of non-mangrove fungi, and the results suggest that there are differences in genetic information among fungi in different habitats.

Identification of novel β-mannan- and β-glucan-binding modules: evidence for a superfamily of carbohydrate-binding modules

2001 ◽  
Vol 356 (3) ◽  
pp. 791-798 ◽  
Author(s):  
Anwar SUNNA ◽  
Moreland D. GIBBS ◽  
Peter L. BERGQUIST
Keyword(s):  
Secondary Structure ◽  
Protein Secondary Structure ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Experimental Characterization ◽  
Carbohydrate Polymers ◽  
New Family ◽  
Carbohydrate Binding Modules ◽  
Long Chain

Many glycoside hydrolases, which degrade long-chain carbohydrate polymers, possess distinct catalytic modules and non-catalytic carbohydrate-binding modules (CBMs). On the basis of conserved protein secondary structure, we describe here the identification and experimental characterization of novel type of mannanase-associated mannan-binding module and also characterization of two CBM family 4 laminarinase-associated β-glucan-binding modules. These modules are predicted to belong to a superfamily of CBMs which include families 4, 16, 17, 22 and a proposed new family, family 27.

scholarly journals A unique combination of glycoside hydrolases in Streptococcus suis specifically and sequentially acts on host-derived αGal-epitope glycans

2020 ◽  
Vol 295 (31) ◽  
pp. 10638-10652
Author(s):  
Ping Chen ◽  
Ran Liu ◽  
Mengmeng Huang ◽  
Jinlu Zhu ◽  
Dong Wei ◽  
...  
Keyword(s):  
Wide Spectrum ◽  
Streptococcus Suis ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Unique Combination ◽  
Enzymatic Assays ◽  
Tracheal Epithelial Cells ◽  
Carbohydrate Binding Modules ◽  
Host Signaling ◽  
Important Host

Infections by many bacterial pathogens rely on their ability to degrade host glycans by producing glycoside hydrolases (GHs). Here, we discovered a conserved multifunctional GH, SsGalNagA, containing a unique combination of two family 32 carbohydrate-binding modules (CBM), a GH16 domain and a GH20 domain, in the zoonotic pathogen Streptococcus suis 05ZYH33. Enzymatic assays revealed that the SsCBM-GH16 domain displays endo-(β1,4)-galactosidase activity specifically toward the host-derived αGal epitope Gal(α1,3)Gal(β1,4)Glc(NAc)-R, whereas the SsGH20 domain has a wide spectrum of exo-β-N-acetylhexosaminidase activities, including exo-(β1,3)-N-acetylglucosaminidase activity, and employs this activity to act in tandem with SsCBM-GH16 on the αGal-epitope glycan. Further, we found that the CBM32 domain adjacent to the SsGH16 domain is indispensable for SsGH16 catalytic activity. Surface plasmon resonance experiments uncovered that both CBM32 domains specifically bind to αGal-epitope glycan, and together they had a KD of 3.5 mm toward a pentasaccharide αGal-epitope glycan. Cell-binding and αGal epitope removal assays revealed that SsGalNagA efficiently binds to both swine erythrocytes and tracheal epithelial cells and removes the αGal epitope from these cells, suggesting that SsGalNagA functions in nutrient acquisition or alters host signaling in S. suis. Both binding and removal activities were blocked by an αGal-epitope glycan. SsGalNagA is the first enzyme reported to sequentially act on a glycan containing the αGal epitope. These findings shed detailed light on the evolution of GHs and an important host-pathogen interaction.

scholarly journals Enzymatic Adaptation of Bifidobacterium bifidum to Host Glycans, Viewed from Glycoside Hydrolyases and Carbohydrate-Binding Modules

2020 ◽  
Vol 8 (4) ◽  
pp. 481 ◽  
Author(s):  
Toshihiko Katoh ◽  
Miriam N. Ojima ◽  
Mikiyasu Sakanaka ◽  
Hisashi Ashida ◽  
Aina Gotoh ◽  
...  
Keyword(s):  
Carbon Sources ◽  
Bifidobacterium Bifidum ◽  
Glycoside Hydrolases ◽  
Altruistic Behavior ◽  
Carbohydrate Binding ◽  
Human Gut ◽  
Genetic Characteristics ◽  
Beneficial Effects ◽  
Carbohydrate Binding Modules ◽  
Enzymatic Machinery

Certain species of the genus Bifidobacterium represent human symbionts. Many studies have shown that the establishment of symbiosis with such bifidobacterial species confers various beneficial effects on human health. Among the more than ten (sub)species of human gut-associated Bifidobacterium that have significantly varied genetic characteristics at the species level, Bifidobacterium bifidum is unique in that it is found in the intestines of a wide age group, ranging from infants to adults. This species is likely to have adapted to efficiently degrade host-derived carbohydrate chains, such as human milk oligosaccharides (HMOs) and mucin O-glycans, which enabled the longitudinal colonization of intestines. The ability of this species to assimilate various host glycans can be attributed to the possession of an adequate set of extracellular glycoside hydrolases (GHs). Importantly, the polypeptides of those glycosidases frequently contain carbohydrate-binding modules (CBMs) with deduced affinities to the target glycans, which is also a distinct characteristic of this species among members of human gut-associated bifidobacteria. This review firstly describes the prevalence and distribution of B. bifidum in the human gut and then explains the enzymatic machinery that B. bifidum has developed for host glycan degradation by referring to the functions of GHs and CBMs. Finally, we show the data of co-culture experiments using host-derived glycans as carbon sources, which underpin the interesting altruistic behavior of this species as a cross-feeder.

scholarly journals Unique N-glycosylation of a recombinant exo-inulinase from Kluyveromyces cicerisporus and its effect on enzymatic activity and thermostability

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Junyan Ma ◽  
Qian Li ◽  
Haidong Tan ◽  
Hao Jiang ◽  
Kuikui Li ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Jerusalem Artichoke ◽  
Fine Tuning ◽  
Substrate Affinity ◽  
Carbohydrate Binding ◽  
Secondary Structure Analysis ◽  
C Terminus ◽  
Glycosylation Sites ◽  
Carbohydrate Binding Modules

Abstract Background Inulinase can hydrolyze polyfructan into high-fructose syrups and fructoligosaccharides, which are widely used in food, the medical industry and the biorefinery of Jerusalem artichoke. In the present study, a recombinant exo-inulinase (rKcINU1), derived from Kluyveromyces cicerisporus CBS4857, was proven as an N-linked glycoprotein, and the removal of N-linked glycan chains led to reduced activity. Results Five N-glycosylation sites with variable high mannose-type oligosaccharides (Man3–9GlcNAc2) were confirmed in the rKcINU1. The structural modeling showed that all five glycosylation sites (Asn-362, Asn-370, Asn-399, Asn-467 and Asn-526) were located at the C-terminus β-sandwich domain, which has been proven to be more conducive to the occurrence of glycosylation modification than the N-terminus domain. Single-site N-glycosylation mutants with Asn substituted by Gln were obtained, and the Mut with all five N-glycosylation sites removed was constructed, which resulted in the loss of all enzyme activity. Interestingly, the N362Q led to an 18% increase in the specific activity against inulin, while a significant decrease in thermostability (2.91 °C decrease in Tm) occurred, and other single mutations resulted in the decrease in the specific activity to various extents, among which N467Q demonstrated the lowest enzyme activity. Conclusion The increased enzyme activity in N362Q, combined with thermostability testing, 3D modeling, kinetics data and secondary structure analysis, implied that the N-linked glycan chains at the Asn-362 position functioned negatively, mainly as a type of steric hindrance toward its adjacent N-glycans to bring rigidity. Meanwhile, the N-glycosylation at the other four sites positively regulated enzyme activity caused by altered substrate affinity by means of fine-tuning the β-sandwich domain configuration. This may have facilitated the capture and transfer of substrates to the enzyme active cavity, in a manner quite similar to that of carbohydrate binding modules (CBMs), i.e. the chains endowed the β-sandwich domain with the functions of CBM. This study discovered a unique C-terminal sequence which is more favorable to glycosylation, thereby casting a novel view for glycoengineering of enzymes from fungi via redesigning the amino acid sequence at the C-terminal domain, so as to optimize the enzymatic properties.

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