Carbohydrate-binding architecture of the multi-modular α-1,6-glucosyltransferase from Paenibacillus sp. 598K, which produces α-1,6-glucosyl-α-glucosaccharides from starch

2017 ◽  
Vol 474 (16) ◽  
pp. 2763-2778 ◽  
Author(s):  
Zui Fujimoto ◽  
Nobuhiro Suzuki ◽  
Naomi Kishine ◽  
Hitomi Ichinose ◽  
Mitsuru Momma ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Binding Mode ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Surface Binding ◽  
Glucose Binding ◽  
Carbohydrate Binding Modules ◽  
Paenibacillus Sp ◽  
Binding Cleft ◽  
Catalytic Cleft

Paenibacillus sp. 598K α-1,6-glucosyltransferase (Ps6TG31A), a member of glycoside hydrolase family 31, catalyzes exo-α-glucohydrolysis and transglucosylation and produces α-1,6-glucosyl-α-glucosaccharides from α-glucan via its disproportionation activity. The crystal structure of Ps6TG31A was determined by an anomalous dispersion method using a terbium derivative. The monomeric Ps6TG31A consisted of one catalytic (β/α)8-barrel domain and six small domains, one on the N-terminal and five on the C-terminal side. The structures of the enzyme complexed with maltohexaose, isomaltohexaose, and acarbose demonstrated that the ligands were observed in the catalytic cleft and the sugar-binding sites of four β-domains. The catalytic site was structured by a glucose-binding pocket and an aglycon-binding cleft built by two sidewalls. The bound acarbose was located with its non-reducing end pseudosugar docked in the pocket, and the other moieties along one sidewall serving three subsites for the α-1,4-glucan. The bound isomaltooligosaccharide was found on the opposite sidewall, which provided the space for the acceptor molecule to be positioned for attack of the catalytic intermediate covalent complex during transglucosylation. The N-terminal domain recognized the α-1,4-glucan in a surface-binding mode. Two of the five C-terminal domains belong to the carbohydrate-binding modules family 35 and one to family 61. The sugar complex structures indicated that the first family 35 module preferred α-1,6-glucan, whereas the second family 35 module and family 61 module preferred α-1,4-glucan. Ps6TG31A appears to have enhanced transglucosylation activity facilitated by its carbohydrate-binding modules and substrate-binding cleft that positions the substrate and acceptor sugar for the transglucosylation.

scholarly journals Cell Surface Xylanases of the Glycoside Hydrolase Family 10 Are Essential for Xylan Utilization by Paenibacillus sp. W-61 as Generators of Xylo-Oligosaccharide Inducers for the Xylanase Genes

2010 ◽  
Vol 192 (8) ◽  
pp. 2210-2219 ◽  
Author(s):  
Mutsumi Fukuda ◽  
Seiji Watanabe ◽  
Shigeki Yoshida ◽  
Hiroya Itoh ◽  
Yoshifumi Itoh ◽  
...  
Keyword(s):  
Cell Surface ◽  
Glycoside Hydrolase ◽  
Extracellular Enzymes ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Immunogold Staining ◽  
Reverse Transcription Pcr ◽  
Carbohydrate Binding Modules ◽  
Paenibacillus Sp ◽  
Entire Cell

ABSTRACT Paenibacillus sp. W-61 is capable of utilizing water-insoluble xylan for carbon and energy sources and has three xylanase genes, xyn1, xyn3, and xyn5. Xyn1, Xyn3, and Xyn5 are extracellular enzymes of the glycoside hydrolase (GH) families 11, 30, and 10, respectively. Xyn5 contains several domains including those of carbohydrate-binding modules (CBMs) similar to a surface-layer homologous (SLH) protein. This study focused on the role of Xyn5, localized on the cell surface, in water-insoluble xylan utilization. Electron microscopy using immunogold staining revealed Xyn5 clusters over the entire cell surface. Xyn5 was bound to cell wall fractions through its SLH domain. A Δxyn5 mutant grew poorly and produced minimal amounts of Xyn1 and Xyn3 on water-insoluble xylan. A Xyn5 mutant lacking the SLH domain (Xyn5ΔSLH) grew poorly, secreting Xyn5ΔSLH into the medium and producing minimal Xyn1 and Xyn3 on water-insoluble xylan. A mutant with an intact xyn5 produced Xyn5 on the cell surface, grew normally, and actively synthesized Xyn1 and Xyn3 on water-insoluble xylan. Quantitative reverse transcription-PCR showed that xylobiose, generated from water-insoluble xylan decomposition by Xyn5, is the most active inducer for xyn1 and xyn3. Luciferase assays using a Xyn5-luciferase fusion protein suggested that xylotriose is the best inducer for xyn5. The cell surface Xyn5 appears to play two essential roles in water-insoluble xylan utilization: (i) generation of the xylo-oligosaccharide inducers of all the xyn genes from water-insoluble xylan and (ii) attachment of the cells to the substrate so that the generated inducers can be immediately taken up by cells to activate expression of the xyn system.

scholarly journals Deamidation and glycation of a Bacillus licheniformis α-amylase during industrial fermentation can improve detergent wash performance

Amylase ◽  
2021 ◽  
Vol 5 (1) ◽  
pp. 38-49
Author(s):  
Connie Pontoppidan ◽  
Svend G. Kaasgaard ◽  
Carsten P. Sønksen ◽  
Carsten Andersen ◽  
Birte Svensson
Keyword(s):  
Bacillus Licheniformis ◽  
Mutational Analysis ◽  
Peptide Mapping ◽  
Substrate Binding ◽  
Carbohydrate Binding ◽  
Surface Binding ◽  
Lysine Residues ◽  
Tandem Mass Spectrometric ◽  
Binding Cleft ◽  
Household Detergents

Abstract The industrial thermostable Bacillus licheniformis α-amylase (BLA) has wide applications, including in household detergents, and efforts to improve its performance are continuously ongoing. BLA during the industrial production is deamidated and glycated resulting in multiple forms with different isoelectric points. Forty modified positions were identified by tandem mass spectrometric peptide mapping of BLA forms separated by isoelectric focusing. These modified 12 asparagine, 9 glutamine, 8 arginine and 11 lysine residues are mostly situated on the enzyme surface and several belong to regions involved in stability, activity and carbohydrate binding. Eight residues presumed to interact with starch at the active site and surface binding sites (SBSs) were subjected to mutational analysis. Five mutants mimicking deamidation (N→D, Q→E) at the substrate binding cleft showed moderate to no effect on thermostability and k cat and K M for maltoheptaose and amylose. Notably, the mutations improved laundry wash efficiency in detergents at pH 8.5 and 10.0. Replacing three reducing sugar reactive side chains (K→M, R→L) at a distant substrate binding region and two SBSs enhanced wash performance especially in liquid detergent at pH 8.5, slightly improved enzymatic activity and maintained thermostability. Wash performance was most improved (5-fold) for the N265D mutant near substrate binding subsite +3.

scholarly journals Cloning and Characterization of Two Xyloglucanases from Paenibacillus sp. Strain KM21

2005 ◽  
Vol 71 (12) ◽  
pp. 7670-7678 ◽  
Author(s):  
Katsuro Yaoi ◽  
Tomonori Nakai ◽  
Yoshiro Kameda ◽  
Ayako Hiyoshi ◽  
Yasushi Mitsuishi
Keyword(s):  
Glycoside Hydrolase ◽  
Amino Acid Sequences ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
C Terminus ◽  
Paenibacillus Sp ◽  
Genes Encoding ◽  
Molecular Masses

ABSTRACT Two xyloglucan-specific endo-β-1,4-glucanases (xyloglucanases [XEGs]), XEG5 and XEG74, with molecular masses of 40 kDa and 105 kDa, respectively, were isolated from the gram-positive bacterium Paenibacillus sp. strain KM21, which degrades tamarind seed xyloglucan. The genes encoding these XEGs were cloned and sequenced. Based on their amino acid sequences, the catalytic domains of XEG5 and XEG74 were classified in the glycoside hydrolase families 5 and 74, respectively. XEG5 is the first xyloglucanase belonging to glycoside hydrolase family 5. XEG5 lacks a carbohydrate-binding module, while XEG74 has an X2 module and a family 3 type carbohydrate-binding module at its C terminus. The two XEGs were expressed in Escherichia coli, and recombinant forms of the enzymes were purified and characterized. Both XEGs had endoglucanase active only toward xyloglucan and not toward Avicel, carboxymethylcellulose, barley β-1,3/1,4-glucan, or xylan. XEG5 is a typical endo-type enzyme that randomly cleaves the xyloglucan main chain, while XEG74 has dual endo- and exo-mode activities or processive endo-mode activity. XEG5 digested the xyloglucan oligosaccharide XXXGXXXG to produce XXXG, whereas XEG74 digestion of XXXGXXXG resulted in XXX, XXXG, and GXXXG, suggesting that this enzyme cleaves the glycosidic bond of unbranched Glc residues. Analyses using various oligosaccharide structures revealed that unique structures of xyloglucan oligosaccharides can be prepared with XEG74.

scholarly journals Spatially remote motifs cooperatively affect substrate preference of a ruminal GH26-type endo-β-1,4-mannanase

2020 ◽  
Vol 295 (15) ◽  
pp. 5012-5021 ◽  
Author(s):  
Fernanda Mandelli ◽  
Mariana Abrahão Bueno de Morais ◽  
Evandro Antonio de Lima ◽  
Leane Oliveira ◽  
Gabriela Felix Persinoti ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Substrate Binding ◽  
Substrate Recognition ◽  
Systematic Investigation ◽  
Substrate Preference ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules ◽  
Binding Cleft ◽  
Aromatic Cluster ◽  
Shed Light

β-Mannanases from the glycoside hydrolase 26 (GH26) family are retaining hydrolases that are active on complex heteromannans and whose genes are abundant in rumen metagenomes and metatranscriptomes. These enzymes can exhibit distinct modes of substrate recognition and are often fused to carbohydrate-binding modules (CBMs), resulting in a molecular puzzle of mechanisms governing substrate preference and mode of action that has not yet been pieced together. In this study, we recovered a novel GH26 enzyme with a CBM35 module linked to its N terminus (CrMan26) from a cattle rumen metatranscriptome. CrMan26 exhibited a preference for galactomannan as substrate and the crystal structure of the full-length protein at 1.85 Å resolution revealed a unique orientation of the ancillary domain relative to the catalytic interface, strategically positioning a surface aromatic cluster of the ancillary domain as an extension of the substrate-binding cleft, contributing to galactomannan preference. Moreover, systematic investigation of nonconserved residues in the catalytic interface unveiled that residues Tyr195 (−3 subsite) and Trp234 (−5 subsite) from distal negative subsites have a key role in galactomannan preference. These results indicate a novel and complex mechanism for substrate recognition involving spatially remote motifs, distal negative subsites from the catalytic domain, and a surface-associated aromatic cluster from the ancillary domain. These findings expand our molecular understanding of the mechanisms of substrate binding and recognition in the GH26 family and shed light on how some CBMs and their respective orientation can contribute to substrate preference.

scholarly journals Comparative Analyses of Two Thermophilic Enzymes Exhibiting both β-1,4 Mannosidic and β-1,4 Glucosidic Cleavage Activities from Caldanaerobius polysaccharolyticus

2010 ◽  
Vol 192 (16) ◽  
pp. 4111-4121 ◽  
Author(s):  
Yejun Han ◽  
Dylan Dodd ◽  
Charles W. Hespen ◽  
Samuel Ohene-Adjei ◽  
Charles M. Schroeder ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Biochemical Properties ◽  
Degree Of Polymerization ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Biofuel Industry ◽  
Carbohydrate Binding Modules ◽  
Mannanase Activity ◽  
Hydrolysis Of ◽  
Derivatives Of

ABSTRACT The hydrolysis of polysaccharides containing mannan requires endo-1,4-β-mannanase and 1,4-β-mannosidase activities. In the current report, the biochemical properties of two endo-β-1,4-mannanases (Man5A and Man5B) from Caldanaerobius polysaccharolyticus were studied. Man5A is composed of an N-terminal signal peptide (SP), a catalytic domain, two carbohydrate-binding modules (CBMs), and three surface layer homology (SLH) repeats, whereas Man5B lacks the SP, CBMs, and SLH repeats. To gain insights into how the two glycoside hydrolase family 5 (GH5) enzymes may aid the bacterium in energy acquisition and also the potential application of the two enzymes in the biofuel industry, two derivatives of Man5A (Man5A-TM1 [TM1 stands for truncational mutant 1], which lacks the SP and SLH repeats, and Man5A-TM2, which lacks the SP, CBMs, and SLH repeats) and the wild-type Man5B were biochemically analyzed. The Man5A derivatives displayed endo-1,4-β-mannanase and endo-1,4-β-glucanase activities and hydrolyzed oligosaccharides with a degree of polymerization (DP) of 4 or higher. Man5B exhibited endo-1,4-β-mannanase activity and little endo-1,4-β-glucanase activity; however, this enzyme also exhibited 1,4-β-mannosidase and cellodextrinase activities. Man5A-TM1, compared to either Man5A-TM2 or Man5B, had higher catalytic activity with soluble and insoluble polysaccharides, indicating that the CBMs enhance catalysis of Man5A. Furthermore, Man5A-TM1 acted synergistically with Man5B in the hydrolysis of β-mannan and carboxymethyl cellulose. The versatility of the two enzymes, therefore, makes them a resource for depolymerization of mannan-containing polysaccharides in the biofuel industry. Furthermore, on the basis of the biochemical and genomic data, a molecular mechanism for utilization of mannan-containing nutrients by C. polysaccharolyticus is proposed.

scholarly journals Crystallization and preliminary crystallographic studies of a novel noncatalytic carbohydrate-binding module from theRuminococcus flavefacienscellulosome

2015 ◽  
Vol 71 (1) ◽  
pp. 45-48 ◽  
Author(s):  
Immacolata Venditto ◽  
Arun Goyal ◽  
Andrew Thompson ◽  
Luis M. A. Ferreira ◽  
Carlos M. G. A. Fontes ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Cellulolytic Bacterium ◽  
Modular Architecture ◽  
Carbohydrate Binding Modules ◽  
Fundamental Biological Process

Microbial degradation of the plant cell wall is a fundamental biological process with considerable industrial importance. Hydrolysis of recalcitrant polysaccharides is orchestrated by a large repertoire of carbohydrate-active enzymes that display a modular architecture in which a catalytic domain is connectedvialinker sequences to one or more noncatalytic carbohydrate-binding modules (CBMs). CBMs direct the appended catalytic modules to their target substrates, thus potentiating catalysis. The genome of the most abundant ruminal cellulolytic bacterium,Ruminococcus flavefaciensstrain FD-1, provides an opportunity to discover novel cellulosomal proteins involved in plant cell-wall deconstruction. It encodes a modular protein comprising a glycoside hydrolase family 9 catalytic module (GH9) linked to two unclassified tandemly repeated CBMs (termed CBM-Rf6A and CBM-Rf6B) and a C-terminal dockerin. The novel CBM-Rf6A from this protein has been crystallized and data were processed for the native and a selenomethionine derivative to 1.75 and 1.5 Å resolution, respectively. The crystals belonged to orthorhombic and cubic space groups, respectively. The structure was solved by a single-wavelength anomalous dispersion experiment using theCCP4 program suite andSHELXC/D/E.

Analysis of surface binding sites (SBSs) in carbohydrate active enzymes with focus on glycoside hydrolase families 13 and 77 — a mini-review

Biologia ◽  
2014 ◽  
Vol 69 (6) ◽  
Author(s):  
Darrell Cockburn ◽  
Casper Wilkens ◽  
Christian Ruzanski ◽  
Susan Andersen ◽  
Jonas Willum Nielsen ◽  
...  
Keyword(s):  
Binding Sites ◽  
Carbohydrate Binding ◽  
Surface Binding ◽  
Binding Studies ◽  
Carbohydrate Active Enzymes ◽  
Enzyme Active Site ◽  
Functional Roles ◽  
Carbohydrate Binding Modules ◽  
The Many ◽  
Key Residues

AbstractSurface binding sites (SBSs) interact with carbohydrates outside of the enzyme active site. They are frequently situated on catalytic domains and are distinct from carbohydrate binding modules (CBMs). SBSs are found in a variety of enzymes and often seen in crystal structures. Notably about half of the > 45 enzymes (in 17 GH and two GT families) with an identified SBS are from GH13 and a few from GH77, both belonging to clan GH-H of carbohydrate active enzymes. The many enzymes of GH13 with SBSs provide an opportunity to analyse their distribution within this very large and diverse family. SBS containing enzymes in GH13 are spread among 15 subfamilies (two were not assigned a subfamily). Comparison of these SBSs reveals a complex evolutionary history with evidence of conservation of key residues and/or structural location between some SBSs, while others are found at entirely distinct structural locations, suggesting convergent evolution. An array of investigations of the two SBSs in barley α-amylase demonstrated they play different functional roles in binding and degradation of polysaccharides. MalQ from Escherichia coli is an α-1,4-glucanotransferase of GH77, a family that is known to have at least one member that contains an SBS. Whereas MalQ is a single domain enzyme lacking CBMs, its plant orthologue DPE2 contains two N-terminal CBM20s. Surface plasmon resonance binding studies showed that MalQ and DPE2 have a similar affinity for β-cyclodextrin and that MalQ binds malto-oligosaccharides of >DP4 at a second site in competition with β-cyclodextrin yielding a stoichiometry >1. This suggests that MalQ may have an SBS, though its structural location remains unknown.

scholarly journals Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

2012 ◽  
Vol 78 (7) ◽  
pp. 2230-2240 ◽  
Author(s):  
Xiaoyun Su ◽  
Roderick I. Mackie ◽  
Isaac K. O. Cann
Keyword(s):  
Catalytic Efficiency ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Wild Type ◽  
Ph Optimum ◽  
Content Type ◽  
Caldicellulosiruptor Bescii ◽  
Type Protein ◽  
Wild Type Protein ◽  
Carbohydrate Binding Modules

ABSTRACTThermophilic cellulases and hemicellulases are of significant interest to the biofuel industry due to their perceived advantages over their mesophilic counterparts. We describe here biochemical and mutational analyses ofCaldicellulosiruptor besciiCel9B/Man5A (CbCel9B/Man5A), a highly thermophilic enzyme. As one of the highly secreted proteins ofC. bescii, the enzyme is likely to be critical to nutrient acquisition by the bacterium. CbCel9B/Man5A is a modular protein composed of three carbohydrate-binding modules flanked at the N terminus and the C terminus by a glycoside hydrolase family 9 (GH9) module and a GH5 module, respectively. Based on truncational analysis of the polypeptide, the cellulase and mannanase activities within CbCel9B/Man5A were assigned to the N- and C-terminal modules, respectively. CbCel9B/Man5A and its truncational mutants, in general, exhibited a pH optimum of ∼5.5 and a temperature optimum of 85°C. However, at this temperature, thermostability was very low. After 24 h of incubation at 75°C, the wild-type protein maintained 43% activity, whereas a truncated mutant, TM1, maintained 75% activity. The catalytic efficiency with phosphoric acid swollen cellulose as a substrate for the wild-type protein was 7.2 s−1ml/mg, and deleting the GH5 module led to a mutant (TM1) with a 2-fold increase in this kinetic parameter. Deletion of the GH9 module also increased the apparentkcatof the truncated mutant TM5 on several mannan-based substrates; however, a concomitant increase in theKmled to a decrease in the catalytic efficiencies on all substrates. These observations lead us to postulate that the two catalytic activities are coupled in the polypeptide.

scholarly journals Expression and Characterization of a Bifidobacterium adolescentis Beta-Mannanase Carrying Mannan-Binding and Cell Association Motifs

2012 ◽  
Vol 79 (1) ◽  
pp. 133-140 ◽  
Author(s):  
Evelina Kulcinskaja ◽  
Anna Rosengren ◽  
Romany Ibrahim ◽  
Katarína Kolenová ◽  
Henrik Stålbrand
Keyword(s):  
Signal Peptide ◽  
High Performance ◽  
Guar Gum ◽  
Transmembrane Helix ◽  
Exchange Chromatography ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Bifidobacterium Adolescentis ◽  
Content Type ◽  
Carbohydrate Binding Modules

ABSTRACTThe gene encoding β-mannanase (EC 3.2.1.78) BaMan26A from the bacteriumBifidobacterium adolescentis(living in the human gut) was cloned and the gene product characterized. The enzyme was found to be modular and to contain a putative signal peptide. It possesses a catalytic module of the glycoside hydrolase family 26, a predicted immunoglobulin-like module, and two putative carbohydrate-binding modules (CBMs) of family 23. The enzyme is likely cell attached either by the sortase mechanism (LPXTG motif) or via a C-terminal transmembrane helix. The gene was expressed inEscherichia coliwithout the native signal peptide or the cell anchor. Two variants were made: one containing all four modules, designated BaMan26A-101K, and one truncated before the CBMs, designated BaMan26A-53K. BaMan26A-101K, which contains the CBMs, showed an affinity to carob galactomannan having a dissociation constant of 0.34 μM (8.8 mg/liter), whereas BaMan26A-53K did not bind, showing that at least one of the putative CBMs of family 23 is mannan binding. For BaMan26A-53K,kcatwas determined to be 444 s−1andKm21.3 g/liter using carob galactomannan as the substrate at the optimal pH of 5.3. Both of the enzyme variants hydrolyzed konjac glucomannan, as well as carob and guar gum galactomannans to a mixture of oligosaccharides. The dominant product from ivory nut mannan was found to be mannotriose. Mannobiose and mannotetraose were produced to a lesser extent, as shown by high-performance anion-exchange chromatography. Mannobiose was not hydrolyzed, and mannotriose was hydrolyzed at a significantly lower rate than the longer oligosaccharides.

Sign in / Sign up

Export Citation Format

Share Document