Functional Analysis of a Novel β-(1,3)-Glucanase from Corallococcus sp. Strain EGB Containing a Fascin-Like Module

ABSTRACT A novel β-(1,3)-glucanase gene designated lamC, cloned from Corallococcus sp. strain EGB, contains a fascin-like module and a glycoside hydrolase family 16 (GH16) catalytic module. LamC displays broad hydrolytic activity toward various polysaccharides. Analysis of the hydrolytic products revealed that LamC is an exo-acting enzyme on β-(1,3)(1,3)- and β-(1,6)-linked glucan substrates and an endo-acting enzyme on β-(1,4)-linked glucan and xylan substrates. Site-directed mutagenesis of conserved catalytic Glu residues (E304A and E309A) demonstrated that these activities were derived from the same active site. Excision of the fascin-like module resulted in decreased activity toward β-(1,3)(1,3)-linked glucans. The carbohydrate-binding assay showed that the fascin-like module was a novel β-(1,3)-linked glucan-binding module. The functional characterization of the fascin-like module and catalytic module will help us better understand these enzymes and modules. IMPORTANCE In this report of a bacterial β-(1,3)(1,3)-glucanase containing a fascin-like module, we reveal the β-(1,3)(1,3)-glucan-binding function of the fascin-like module present in the N terminus of LamC. LamC displays exo-β-(1,3)/(1,6)-glucanase and endo-β-(1,4)-glucanase/xylanase activities with a single catalytic domain. Thus, LamC was identified as a novel member of the GH16 family.

Download Full-text

Functional Characterization of Carbohydrate-Binding Modules in a New Alginate Lyase, TsAly7B, from Thalassomonas sp. LD5

Marine Drugs ◽

10.3390/md18010025 ◽

2019 ◽

Vol 18 (1) ◽

pp. 25 ◽

Cited By ~ 4

Author(s):

Zhelun Zhang ◽

Luyao Tang ◽

Mengmeng Bao ◽

Zhigang Liu ◽

Wengong Yu ◽

...

Keyword(s):

Specific Activity ◽

Biological Activities ◽

Functional Characterization ◽

Alginate Lyase ◽

Carbohydrate Binding ◽

Enzyme Degradation ◽

Carbohydrate Binding Modules ◽

Catalytic Module ◽

Alginate Lyases

Alginate lyases degrade alginate into oligosaccharides, of which the biological activities have vital roles in various fields. Some alginate lyases contain one or more carbohydrate-binding modules (CBMs), which assist the function of the catalytic modules. However, the precise function of CBMs in alginate lyases has yet to be fully elucidated. We have identified a new multi-domain alginate lyase, TsAly7B, in the marine bacterium Thalassomonas sp. LD5. This novel lyase contains an N-terminal CBM9, an internal CBM32, and a C-terminal polysaccharide lyase family 7 (PL7) catalytic module. To investigate the specific function of each of these CBMs, we expressed and characterized the full-length TsAly7B and three truncated mutants: TM1 (CBM32-PL7), TM2 (CBM9-PL7), and TM3 (PL7 catalytic module). CBM9 and CBM32 could enhance the degradation of alginate. Notably, the specific activity of TM2 was 7.6-fold higher than that of TM3. CBM32 enhanced the resistance of the catalytic module to high temperatures. In addition, a combination of CBM9 and CBM32 showed enhanced thermostability when incubated at 80 °C for 1 h. This is the first report that finds CBM9 can significantly improve the ability of enzyme degradation. Our findings provide new insight into the interrelationships of tandem CBMs and alginate lyases and other polysaccharide-degrading enzymes, which may inspire CBM fusion strategies.

Download Full-text

S-Layer Homology Domain Proteins Csac_0678 and Csac_2722 Are Implicated in Plant Polysaccharide Deconstruction by the Extremely Thermophilic Bacterium Caldicellulosiruptor saccharolyticus

Applied and Environmental Microbiology ◽

10.1128/aem.07031-11 ◽

2011 ◽

Vol 78 (3) ◽

pp. 768-777 ◽

Cited By ~ 43

Author(s):

Inci Ozdemir ◽

Sara E. Blumer-Schuette ◽

Robert M. Kelly

Keyword(s):

Cellular Localization ◽

Catalytic Domain ◽

Plant Biomass ◽

Thermophilic Bacteria ◽

Biochemical Properties ◽

Crystalline Cellulose ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Caldicellulosiruptor Saccharolyticus ◽

Content Type

ABSTRACTThe genusCaldicellulosiruptorcontains extremely thermophilic bacteria that grow on plant polysaccharides. The genomes ofCaldicellulosiruptorspecies reveal certain surface layer homology (SLH) domain proteins that have distinguishing features, pointing to a role in lignocellulose deconstruction. Two of these proteins inCaldicellulosiruptor saccharolyticus(Csac_0678 and Csac_2722) were examined from this perspective. In addition to three contiguous SLH domains, the Csac_0678 gene encodes a glycoside hydrolase family 5 (GH5) catalytic domain and a family 28 carbohydrate-binding module (CBM); orthologs to Csac_0678 could be identified in all genome-sequencedCaldicellulosiruptorspecies. Recombinant Csac_0678 was optimally active at 75°C and pH 5.0, exhibiting both endoglucanase and xylanase activities. SLH domain removal did not impact Csac_0678 GH activity, but deletion of the CBM28 domain eliminated binding to crystalline cellulose and rendered the enzyme inactive on this substrate. Csac_2722 is the largest open reading frame (ORF) in theC. saccharolyticusgenome (predicted molecular mass of 286,516 kDa) and contains two putative sugar-binding domains, two Big4 domains (bacterial domains with an immunoglobulin [Ig]-like fold), and a cadherin-like (Cd) domain. Recombinant Csac_2722, lacking the SLH and Cd domains, bound to cellulose and had detectable carboxymethylcellulose (CMC) hydrolytic activity. Antibodies directed against Csac_0678 and Csac_2722 confirmed that these proteins bound to theC. saccharolyticusS-layer. Their cellular localization and functional biochemical properties indicate roles for Csac_0678 and Csac_2722 in recruitment and hydrolysis of complex polysaccharides and the deconstruction of lignocellulosic biomass. Furthermore, these results suggest that related SLH domain proteins in otherCaldicellulosiruptorgenomes may also be important contributors to plant biomass utilization.

Download Full-text

Influence of a Mannan Binding Family 32 Carbohydrate Binding Module on the Activity of the Appended Mannanase

Applied and Environmental Microbiology ◽

10.1128/aem.07457-11 ◽

2012 ◽

Vol 78 (14) ◽

pp. 4781-4787 ◽

Cited By ~ 23

Author(s):

Kimiya Mizutani ◽

Vânia O. Fernandes ◽

Shuichi Karita ◽

Ana S. Luís ◽

Makiko Sakka ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Guar Gum ◽

Catalytic Domain ◽

Hypothetical Protein ◽

Carbohydrate Binding ◽

Type I ◽

Content Type ◽

Carbohydrate Binding Modules ◽

Catalytic Module ◽

Product Profile

ABSTRACTIn general, cellulases and hemicellulases are modular enzymes in which the catalytic domain is appended to one or more noncatalytic carbohydrate binding modules (CBMs). CBMs, by concentrating the parental enzyme at their target polysaccharide, increase the capacity of the catalytic module to bind the substrate, leading to a potentiation in catalysis.Clostridium thermocellumhypothetical protein Cthe_0821, defined here asC. thermocellumMan5A, is a modular protein comprising an N-terminal signal peptide, a family 5 glycoside hydrolase (GH5) catalytic module, a family 32 CBM (CBM32), and a C-terminal type I dockerin module. Recent proteomic studies revealed that Cthe_0821 is one of the major cellulosomal enzymes whenC. thermocellumis cultured on cellulose. Here we show that the GH5 catalytic module of Cthe_0821 displays endomannanase activity.C. thermocellumMan5A hydrolyzes soluble konjac glucomannan, soluble carob galactomannan, and insoluble ivory nut mannan but does not attack the highly galactosylated mannan from guar gum, suggesting that the enzyme prefers unsubstituted β-1,4-mannoside linkages. The CBM32 ofC. thermocellumMan5A displays a preference for the nonreducing ends of mannooligosaccharides, although the protein module exhibits measurable affinity for the termini of β-1,4-linked glucooligosaccharides such as cellobiose. CBM32 potentiates the activity ofC. thermocellumMan5A against insoluble mannans but has no significant effect on the capacity of the enzyme to hydrolyze soluble galactomannans and glucomannans. The product profile ofC. thermocellumMan5A is affected by the presence of CBM32.

Download Full-text

Biochemical and Structural Characterizations of Two Dictyostelium Cellobiohydrolases from the Amoebozoa Kingdom Reveal a High Level of Conservation between Distant Phylogenetic Trees of Life

Applied and Environmental Microbiology ◽

10.1128/aem.00163-16 ◽

2016 ◽

Vol 82 (11) ◽

pp. 3395-3409 ◽

Cited By ~ 8

Author(s):

Sarah E. Hobdey ◽

Brandon C. Knott ◽

Majid Haddad Momeni ◽

Larry E. Taylor ◽

Anna S. Borisova ◽

...

Keyword(s):

Active Site ◽

Phylogenetic Trees ◽

Plant Cell Wall ◽

Catalytic Domain ◽

Cellulolytic Enzymes ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Industrial Enzymes ◽

Content Type ◽

Social Amoeba

ABSTRACTGlycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) are enzymes commonly employed in plant cell wall degradation across eukaryotic kingdoms of life, as they provide significant hydrolytic potential in cellulose turnover. To date, many fungal GH7 CBHs have been examined, yet many questions regarding structure-activity relationships in these important natural and commercial enzymes remain. Here, we present the crystal structures and a biochemical analysis of two GH7 CBHs from social amoeba:Dictyostelium discoideumCel7A (DdiCel7A) andDictyostelium purpureumCel7A (DpuCel7A).DdiCel7A andDpuCel7A natively consist of a catalytic domain and do not exhibit a carbohydrate-binding module (CBM). The structures ofDdiCel7A andDpuCel7A, resolved to 2.1 Å and 2.7 Å, respectively, are homologous to those of other GH7 CBHs with an enclosed active-site tunnel. Two primary differences between theDictyosteliumCBHs and the archetypal model GH7 CBH,Trichoderma reeseiCel7A (TreCel7A), occur near the hydrolytic active site and the product-binding sites. To compare the activities of these enzymes with the activity ofTreCel7A, the family 1TreCel7A CBM and linker were added to the C terminus of each of theDictyosteliumenzymes, creatingDdiCel7ACBMandDpuCel7ACBM, which were recombinantly expressed inT. reesei.DdiCel7ACBMandDpuCel7ACBMhydrolyzed Avicel, pretreated corn stover, and phosphoric acid-swollen cellulose as efficiently asTreCel7A when hydrolysis was compared at their temperature optima. TheKiof cellobiose was significantly higher forDdiCel7ACBMandDpuCel7ACBMthan forTreCel7A: 205, 130, and 29 μM, respectively. Taken together, the present study highlights the remarkable degree of conservation of the activity of these key natural and industrial enzymes across quite distant phylogenetic trees of life.IMPORTANCEGH7 CBHs are among the most important cellulolytic enzymes both in nature and for emerging industrial applications for cellulose breakdown. Understanding the diversity of these key industrial enzymes is critical to engineering them for higher levels of activity and greater stability. The present work demonstrates that two GH7 CBHs from social amoeba are surprisingly quite similar in structure and activity to the canonical GH7 CBH from the model biomass-degrading fungusT. reeseiwhen tested under equivalent conditions (with added CBM-linker domains) on an industrially relevant substrate.

Download Full-text

A New Group of Modular Xylanases in Glycoside Hydrolase Family 8 from Marine Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.01785-18 ◽

2018 ◽

Vol 84 (23) ◽

Cited By ~ 2

Author(s):

Xiu-Lan Chen ◽

Fang Zhao ◽

Yong-Sheng Yue ◽

Xi-Ying Zhang ◽

Yu-Zhong Zhang ◽

...

Keyword(s):

Marine Bacteria ◽

Glycoside Hydrolase ◽

Catalytic Domain ◽

Domain Architecture ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Carbohydrate Binding Module ◽

Binding Ability ◽

Content Type ◽

Hydrolase Family

ABSTRACT Xylanases play a crucial role in the degradation of xylan in both terrestrial and marine environments. The endoxylanase XynB from the marine bacterium Glaciecola mesophila KMM 241 is a modular enzyme comprising a long N-terminal domain (NTD) (E44 to T562) with xylan-binding ability and a catalytic domain (CD) (T563 to E912) of glycoside hydrolase family 8 (GH8). In this study, the long NTD is confirmed to contain three different functional regions, which are NTD1 (E44 to D136), NTD2 (Y137 to A193), and NTD3 (L194 to T562). NTD1, mainly composed of eight β-strands, functions as a new type of carbohydrate-binding module (CBM), which has xylan-binding ability but no sequence similarity to any known CBM. NTD2, mainly forming two α-helices, contains one of the α-helices of the catalytic domain's (α/α)6 barrel and therefore is essential for the activity of XynB, although it is far away from the catalytic domain in sequence. NTD3, next to the catalytic domain in sequence, is shown to be helpful in maintaining the thermostability of XynB. Thus, XynB represents a kind of xylanase with a new domain architecture. There are four other predicted glycoside hydrolase sequences with the same domain architecture and high sequence identity (≥80%) with XynB, all of which are from marine bacteria. Phylogenetic analysis shows that XynB and these homologs form a new group in GH8, representing a new class of marine bacterial xylanases. Our results shed light on xylanases, especially marine xylanases. IMPORTANCE Xylanases play a crucial role in natural xylan degradation and have been extensively used in industries such as food processing, animal feed, and kraft pulp biobleaching. Some marine bacteria have been found to secrete xylanases. Characterization of novel xylanases from marine bacteria has significance for both the clarification of xylan degradation mechanisms in the sea and the development of new enzymes for industrial application. With G. mesophila XynB as a representative, this study reveals a new group of the GH8 xylanases from marine bacteria, which have a distinct domain architecture and contain a novel carbohydrate-binding module. Thus, this study offers new knowledge on marine xylanases.

Download Full-text

Processivity and Enzymatic Mode of a Glycoside Hydrolase Family 5 Endoglucanase from Volvariella volvacea

Applied and Environmental Microbiology ◽

10.1128/aem.02725-12 ◽

2012 ◽

Vol 79 (3) ◽

pp. 989-996 ◽

Cited By ~ 38

Author(s):

Fei Zheng ◽

Shaojun Ding

Keyword(s):

Glycoside Hydrolase ◽

Catalytic Domain ◽

Specific Activity ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Volvariella Volvacea ◽

Reaction Conditions ◽

Content Type ◽

C Terminus ◽

Hydrolase Family

ABSTRACTEG1 is a modular glycoside hydrolase family 5 endoglucanase fromVolvariella volvaceaconsisting of an N-terminal carbohydrate-binding module (CBM1) and a catalytic domain (CD). The ratios of soluble to insoluble reducing sugar produced from filter paper after 8 and 24 h of exposure to EG1 were 6.66 and 8.56, respectively, suggesting that it is a processive endoglucanase. Three derivatives of EG1 containing a core domain only or additional CBMs were constructed in order to evaluate the contribution of the CBM to the processivity and enzymatic mode of EG1 under stationary and agitated conditions. All four enzymatic forms exhibited the same mode of action on both soluble and insoluble cellulosic substrates with cellobiose as a main end product. An additional CBM fused at either the N or C terminus reduced specific activity toward soluble and insoluble celluloses under stationary reaction conditions. Deletion of the CBM significantly decreased enzyme processivity. Insertion of an additional CBM also resulted in a dramatic decrease in processivity in enzyme-substrate reaction mixtures incubated for 0.5 h, but this effect was reversed when reactions were allowed to proceed for longer periods (24 h). Further significant differences were observed in the substrate adsorption/desorption patterns of EG1 and enzyme derivatives equipped with an additional CBM under agitated reaction conditions. An additional family 1 CBM improved EG1 processivity on insoluble cellulose under highly agitated conditions. Our data indicate a strong link between high adsorption levels and low desorption levels in the processivity of EG1 and possibly other processive endoglucanses.

Download Full-text

Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

Microorganisms ◽

10.3390/microorganisms9051107 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1107

Author(s):

Wonho Choi ◽

Yoshihiro Yamaguchi ◽

Ji-Young Park ◽

Sang-Hyun Park ◽

Hyeok-Won Lee ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Physiological Function ◽

Functional Characterization ◽

Site Directed Mutagenesis ◽

In Vitro Assays ◽

Cell Growth Inhibition ◽

The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

Download Full-text

Characterization of Glycoside Hydrolase Family 5 Proteins in Schizosaccharomyces pombe

Eukaryotic Cell ◽

10.1128/ec.00187-10 ◽

2010 ◽

Vol 9 (11) ◽

pp. 1650-1660 ◽

Cited By ~ 11

Author(s):

Encarnación Dueñas-Santero ◽

Ana Belén Martín-Cuadrado ◽

Thierry Fontaine ◽

Jean-Paul Latgé ◽

Francisco del Rey ◽

...

Keyword(s):

Cell Wall ◽

Schizosaccharomyces Pombe ◽

Protein Characterization ◽

Wall Material ◽

Glycoside Hydrolase Family ◽

Content Type ◽

Hydrolysis Of ◽

Controlled Hydrolysis ◽

Hydrolase Family

ABSTRACT In yeast, enzymes with β-glucanase activity are thought to be necessary in morphogenetic events that require controlled hydrolysis of the cell wall. Comparison of the sequence of the Saccharomyces cerevisiae exo-β(1,3)-glucanase Exg1 with the Schizosaccharomyces pombe genome allowed the identification of three genes that were named exg1 + (locus SPBC1105.05), exg2 + (SPAC12B10.11), and exg3 + (SPBC2D10.05). The three proteins have different localizations: Exg1 is secreted to the periplasmic space, Exg2 is a membrane protein, and Exg3 is a cytoplasmic protein. Characterization of the biochemical activity of the proteins indicated that Exg1 and Exg3 are active only against β(1,6)-glucans while no activity was detected for Exg2. Interestingly, Exg1 cleaves the glucans with an endohydrolytic mode of action. exg1 + showed periodic expression during the cell cycle, with a maximum coinciding with the septation process, and its expression was dependent on the transcription factor Sep1. The Exg1 protein localizes to the septum region in a pattern that was different from that of the endo-β(1,3)-glucanase Eng1. Overexpression of Exg2 resulted in an increase in cell wall material at the poles and in the septum, but the putative catalytic activity of the protein was not required for this effect.

Download Full-text

N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01310-17 ◽

2017 ◽

Vol 61 (11) ◽

Cited By ~ 6

Author(s):

Stanislav Huszár ◽

Vinayak Singh ◽

Alica Polčicová ◽

Peter Baráth ◽

María Belén Barrio ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Target ◽

Functional Characterization ◽

Drug Candidate ◽

Content Type ◽

Chemical Libraries ◽

Whole Cells ◽

Encoding Gene

ABSTRACT The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

Download Full-text

Functional Characterization of a Novel Promoter Element Required for an Innate Immune Response in Drosophila

Molecular and Cellular Biology ◽

10.1128/mcb.23.22.8272-8281.2003 ◽

2003 ◽

Vol 23 (22) ◽

pp. 8272-8281 ◽

Cited By ~ 21

Author(s):

Hanna Uvell ◽

Ylva Engström

Keyword(s):

Gene Expression ◽

Antimicrobial Peptide ◽

Functional Characterization ◽

Binding Activity ◽

Innate Immune ◽

Site Directed Mutagenesis ◽

Promoter Element ◽

Peptide Gene ◽

Inducible Genes

ABSTRACT Innate immune reactions are crucial processes of metazoans to protect the organism against overgrowth of faster replicating microorganisms. Drosophila melanogaster is a precious model for genetic and molecular studies of the innate immune system. In response to infection, the concerted action of a battery of antimicrobial peptides ensures efficient killing of the microbes. The induced gene expression relies on translocation of the Drosophila Rel transcription factors Relish, Dif, and Dorsal to the nucleus where they bind to κB-like motifs in the promoters of the inducible genes. We have identified another putative promoter element, called region 1 (R1), in a number of antimicrobial peptide genes. Site-directed mutagenesis of the R1 site diminished Cecropin A1 (CecA1) expression in transgenic Drosophila larvae and flies. Infection of flies induced a nuclear R1-binding activity that was unrelated to the κB-binding activity in the same extracts. Although the R1 motif was required for Rel protein-mediated CecA1 expression in cotransfection experiments, our data argue against it being a direct target for the Drosophila Rel proteins. We propose that the R1 and κB motifs are targets for distinct regulatory complexes that act in concert to promote high levels of antimicrobial peptide gene expression in response to infection.

Download Full-text