scholarly journals NMR structures and functional roles of two related chitin-binding domains of a lytic polysaccharide monooxygenase from Cellvibrio japonicus

2021 ◽  
Author(s):  
Eva Madland ◽  
Zarah Forsberg ◽  
Yong Wang ◽  
Kresten Lindorff-Larsen ◽  
Axel Niebisch ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Carbohydrate Binding ◽  
Functional Variation ◽  
Lytic Polysaccharide Monooxygenase ◽  
Functional Roles ◽  
Binding Domains ◽  
Carbohydrate Binding Modules ◽  
Structural And Functional Properties ◽  
Polysaccharide Monooxygenases ◽  
Dynamics Simulations

AbstractAmong the extensive repertoire of carbohydrate-active enzymes, lytic polysaccharide monooxygenases (LPMOs) have a key role in recalcitrant biomass degradation. LPMOs are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides such as cellulose and chitin. Several LPMOs contain carbohydrate-binding modules (CBMs) that are known to promote LPMO efficiency. Still, structural and functional properties of some of these CBMs remain unknown and it is not clear why some LPMOs, like CjLPMO10A from Cellvibrio japonicus, have two CBMs (CjCBM5 and CjCBM73). Here, we studied substrate binding by these two CBMs to shine light on the functional variation, and determined the solution structures of both by NMR, which includes the first structure of a member of the CBM73 family. Chitin-binding experiments and molecular dynamics simulations showed that, while both CBMs bind crystalline chitin with Kd values in the µM range, CjCBM73 has higher affinity than CjCBM5. Furthermore, NMR titration experiments showed that CjCBM5 binds soluble chitohexaose, whereas no binding to soluble chitin was detected for CjCBM73. These functional differences correlated with distinctly different architectures of the substrate-binding surfaces of the two CBMs. Taken together, these results provide insight into natural variation among related chitin-binding CBMs and the possible functional implications of such variation.

scholarly journals Glucoamylase starch-binding domain of Aspergillus niger B1: molecular cloning and functional characterization

2003 ◽  
Vol 372 (3) ◽  
pp. 905-910 ◽  
Author(s):  
Tzur PALDI ◽  
Ilan LEVY ◽  
Oded SHOSEYOV
Keyword(s):  
Aspergillus Niger ◽  
Corn Starch ◽  
Catalytic Domain ◽  
Functional Characterization ◽  
Carbohydrate Binding ◽  
Amylolytic Enzymes ◽  
Binding Domains ◽  
C Terminus ◽  
Carbohydrate Binding Modules ◽  
Modified Starches

Carbohydrate-binding modules (CBMs) are protein domains located within a carbohydrate-active enzyme, with a discrete fold that can be separated from the catalytic domain. Starch-binding domains (SBDs) are CBMs that are usually found at the C-terminus in many amylolytic enzymes. The SBD from Aspergillus niger B1 (CMI CC 324262) was cloned and expressed in Escherichia coli as an independent domain and the recombinant protein was purified on starch. The A. niger B1 SBD was found to be similar to SBD from A. kawachii, A. niger var. awamori and A. shirusami (95–96% identity) and was classified as a member of the CBM family 20. Characterization of SBD binding to starch indicated that it is essentially irreversible and that its affinity to cationic or anionic starch, as well as to potato or corn starch, does not differ significantly. These observations indicate that the fundamental binding area on these starches is essentially the same. Natural and chemically modified starches are among the most useful biopolymers employed in the industry. Our study demonstrates that SBD binds effectively to both anionic and cationic starch.

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 Substrate Binding Effects of Carbohydrate Binding Modules on the Catalytic Activity of a Multifunctional Cellulase

2015 ◽  
Vol 108 (2) ◽  
pp. 225a
Author(s):  
Johnnie A. Walker ◽  
Taichi E. Takasuka ◽  
Kai Deng ◽  
Christopher M. Bianchetti ◽  
Hannah Udell ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Substrate Binding ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules

scholarly journals Investigation of a thermostable multi-domain xylanase-glucuronoyl esterase enzyme from Caldicellulosiruptor kristjanssonii incorporating multiple carbohydrate-binding modules

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Daniel Krska ◽  
Johan Larsbrink
Keyword(s):  
Biochemical Characterization ◽  
Thermostable Enzyme ◽  
Industrial Processes ◽  
Dual Function ◽  
Carbohydrate Binding ◽  
Binding Assays ◽  
Catalytic Domains ◽  
Binding Domains ◽  
Carbohydrate Binding Modules ◽  
Glucuronoyl Esterase

Abstract Background Efficient degradation of lignocellulosic biomass has become a major bottleneck in industrial processes which attempt to use biomass as a carbon source for the production of biofuels and materials. To make the most effective use of the source material, both the hemicellulosic as well as cellulosic parts of the biomass should be targeted, and as such both hemicellulases and cellulases are important enzymes in biorefinery processes. Using thermostable versions of these enzymes can also prove beneficial in biomass degradation, as they can be expected to act faster than mesophilic enzymes and the process can also be improved by lower viscosities at higher temperatures, as well as prevent the introduction of microbial contamination. Results This study presents the investigation of the thermostable, dual-function xylanase-glucuronoyl esterase enzyme CkXyn10C-GE15A from the hyperthermophilic bacterium Caldicellulosiruptor kristjanssonii. Biochemical characterization of the enzyme was performed, including assays for establishing the melting points for the different protein domains, activity assays for the two catalytic domains, as well as binding assays for the multiple carbohydrate-binding domains present in CkXyn10C-GE15A. Although the enzyme domains are naturally linked together, when added separately to biomass, the expected boosting of the xylanase action was not seen. This lack of intramolecular synergy might suggest, together with previous data, that increased xylose release is not the main beneficial trait given by glucuronoyl esterases. Conclusions Due to its thermostability, CkXyn10C-GE15A is a promising candidate for industrial processes, with both catalytic domains exhibiting melting temperatures over 70 °C. Of particular interest is the glucuronoyl esterase domain, as it represents the first studied thermostable enzyme displaying this activity.

The evolutionary origin and possible functional roles of FNIII domains in two Microbacterium aurum B8.A granular starch degrading enzymes, and in other carbohydrate acting enzymes

Amylase ◽  
2017 ◽  
Vol 1 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Vincent Valk ◽  
Rachel M. van der Kaaij ◽  
Lubbert Dijkhuizen
Keyword(s):  
Protein Interactions ◽  
Catalytic Domain ◽  
Substrate Surface ◽  
Carbohydrate Binding ◽  
Protein Protein Interactions ◽  
Functional Roles ◽  
Cazy Database ◽  
Carbohydrate Binding Modules ◽  
Fibronectin Type Iii

AbstractFibronectin type III (FNIII) domains were first identified in the eukaryotic plasma protein fibronectin, where they act as structural spacers or enable protein-protein interactions. Recently we characterized two large and multi-domain amylases in Microbacterium aurum B8.A that both carry multiple FNIII and carbohydrate binding modules (CBMs). The role of (multiple) FNIII domains in such carbohydrate acting enzymes is currently unclear. Four hypothetical functions are considered here: a substrate surface disruption domain, a carbohydrate binding module, as a stable linker, or enabling protein-protein interactions. We performed a phylogenetic analysis of all FNIII domains identified in proteins listed in the CAZy database. These data clearly show that the FNIII domains in eukaryotic and archaeal CAZy proteins are of bacterial origin and also provides examples of interkingdom gene transfer from Bacteria to Archaea and Eucarya. FNIII domains occur in a wide variety of CAZy enzymes acting on many different substrates, suggesting that they have a non-specific role in these proteins. While CBM domains are mostly found at protein termini, FNIII domains are commonly located between other protein domains. FNIII domains in carbohydrate acting enzymes thus may function mainly as stable linkers to allow optimal positioning and/or flexibility of the catalytic domain and other domains, such as CBM.

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 The structure of a glycoside hydrolase 29 family member from a rumen bacterium reveals unique, dual carbohydrate-binding domains

2016 ◽  
Vol 72 (10) ◽  
pp. 750-761 ◽  
Author(s):  
Emma L. Summers ◽  
Christina D. Moon ◽  
Renee Atua ◽  
Vickery L. Arcus
Keyword(s):  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Binding Domain ◽  
Carbohydrate Binding ◽  
Catalytic Core ◽  
Binding Domains ◽  
Carbohydrate Binding Modules ◽  
Tim Barrel ◽  
Domain Arrangement ◽  
Hydrolysis Of

Glycoside hydrolase (GH) family 29 consists solely of α-L-fucosidases. These enzymes catalyse the hydrolysis of glycosidic bonds. Here, the structure of GH29_0940, a protein cloned from metagenomic DNA from the rumen of a cow, has been solved, which reveals a multi-domain arrangement that has only recently been identified in bacterial GH29 enzymes. The microbial species that provided the source of this enzyme is unknown. This enzyme contains a second carbohydrate-binding domain at its C-terminal end in addition to the typical N-terminal catalytic domain and carbohydrate-binding domain arrangement of GH29-family proteins. GH29_0940 is a monomer and its overall structure consists of an N-terminal TIM-barrel-like domain, a central β-sandwich domain and a C-terminal β-sandwich domain. The TIM-barrel-like catalytic domain exhibits a (β/α)8/7arrangement in the core instead of the typical (β/α)8topology, with the `missing' α-helix replaced by a long meandering loop that `closes' the barrel structure and suggests a high degree of structural flexibility in the catalytic core. This feature was also noted in all six other structures of GH29 enzymes that have been deposited in the PDB. Based on sequence and structural similarity, the residues Asp162 and Glu220 are proposed to serve as the catalytic nucleophile and the proton donor, respectively. Like other GH29 enzymes, the GH29_0940 structure shows five strictly conserved residues in the catalytic pocket. The structure shows two glycerol molecules in the active site, which have also been observed in other GH29 structures, suggesting that the enzyme catalyses the hydrolysis of small carbohydrates. The two binding domains are classed as family 32 carbohydrate-binding modules (CBM32). These domains have residues involved in ligand binding in the loop regions at the edge of the β-sandwich. The predicted substrate-binding residues differ between the modules, suggesting that different modules bind to different groups on the substrate(s). Enzymes that possess multiple copies of CBMs are thought to have a complex mechanism of ligand recognition. Defined electron density identifying a long 20-amino-acid hydrophilic loop separating the two CBMs was observed. This suggests that the additional C-terminal domain may have a dynamic range of movement enabled by the loop, allowing a unique mode of action for a GH29 enzyme that has not been identified previously.

scholarly journals The lytic polysaccharide monooxygenase CbpD promotes Pseudomonas aeruginosa virulence in systemic infection

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Fatemeh Askarian ◽  
Satoshi Uchiyama ◽  
Helen Masson ◽  
Henrik Vinther Sørensen ◽  
Ole Golten ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Catalytic Activity ◽  
Inflammatory Responses ◽  
Ex Vivo ◽  
Systemic Infection ◽  
Lytic Polysaccharide Monooxygenase ◽  
Redox Active ◽  
Polysaccharide Monooxygenases ◽  
Dynamics Simulations

AbstractThe recently discovered lytic polysaccharide monooxygenases (LPMOs), which cleave polysaccharides by oxidation, have been associated with bacterial virulence, but supporting functional data is scarce. Here we show that CbpD, the LPMO of Pseudomonas aeruginosa, is a chitin-oxidizing virulence factor that promotes survival of the bacterium in human blood. The catalytic activity of CbpD was promoted by azurin and pyocyanin, two redox-active virulence factors also secreted by P. aeruginosa. Homology modeling, molecular dynamics simulations, and small angle X-ray scattering indicated that CbpD is a monomeric tri-modular enzyme with flexible linkers. Deletion of cbpD rendered P. aeruginosa unable to establish a lethal systemic infection, associated with enhanced bacterial clearance in vivo. CbpD-dependent survival of the wild-type bacterium was not attributable to dampening of pro-inflammatory responses by CbpD ex vivo or in vivo. Rather, we found that CbpD attenuates the terminal complement cascade in human serum. Studies with an active site mutant of CbpD indicated that catalytic activity is crucial for virulence function. Finally, profiling of the bacterial and splenic proteomes showed that the lack of this single enzyme resulted in substantial re-organization of the bacterial and host proteomes. LPMOs similar to CbpD occur in other pathogens and may have similar immune evasive functions.

scholarly journals Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC

2016 ◽  
Vol 113 (37) ◽  
pp. 10358-10363 ◽  
Author(s):  
Hüseyin Ilgü ◽  
Jean-Marc Jeckelmann ◽  
Vytautas Gapsys ◽  
Zöhre Ucurum ◽  
Bert L. de Groot ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Radioligand Binding ◽  
Substrate Binding ◽  
Acid Resistance ◽  
Enteric Bacteria ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Functional Roles ◽  
Dynamics Simulations ◽  
Substrate Binding Sites

Pathogenic enterobacteria need to survive the extreme acidity of the stomach to successfully colonize the human gut. Enteric bacteria circumvent the gastric acid barrier by activating extreme acid-resistance responses, such as the arginine-dependent acid resistance system. In this response, l-arginine is decarboxylated to agmatine, thereby consuming one proton from the cytoplasm. In Escherichia coli, the l-arginine/agmatine antiporter AdiC facilitates the export of agmatine in exchange of l-arginine, thus providing substrates for further removal of protons from the cytoplasm and balancing the intracellular pH. We have solved the crystal structures of wild-type AdiC in the presence and absence of the substrate agmatine at 2.6-Å and 2.2-Å resolution, respectively. The high-resolution structures made possible the identification of crucial water molecules in the substrate-binding sites, unveiling their functional roles for agmatine release and structure stabilization, which was further corroborated by molecular dynamics simulations. Structural analysis combined with site-directed mutagenesis and the scintillation proximity radioligand binding assay improved our understanding of substrate binding and specificity of the wild-type l-arginine/agmatine antiporter AdiC. Finally, we present a potential mechanism for conformational changes of the AdiC transport cycle involved in the release of agmatine into the periplasmic space of E. coli.

scholarly journals A trimodular bacterial enzyme combining hydrolytic activity with oxidative glycosidic bond cleavage efficiently degrades chitin

2020 ◽  
Vol 295 (27) ◽  
pp. 9134-9146 ◽  
Author(s):  
Sophanit Mekasha ◽  
Tina Rise Tuveng ◽  
Fatemeh Askarian ◽  
Swati Choudhary ◽  
Claudia Schmidt-Dannert ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Bond Cleavage ◽  
Hydrolytic Enzymes ◽  
Hydrolytic Activity ◽  
Full Length ◽  
Spatial Proximity ◽  
Carbohydrate Binding ◽  
Lytic Polysaccharide Monooxygenase ◽  
Chitin Binding Domain ◽  
Carbohydrate Binding Modules

Findings from recent studies have indicated that enzymes containing more than one catalytic domain may be particularly powerful in the degradation of recalcitrant polysaccharides such as chitin and cellulose. Some known multicatalytic enzymes contain several glycoside hydrolase domains and one or more carbohydrate-binding modules (CBMs). Here, using bioinformatics and biochemical analyses, we identified an enzyme, Jd1381 from the actinobacterium Jonesia denitrificans, that uniquely combines two different polysaccharide-degrading activities. We found that Jd1381 contains an N-terminal family AA10 lytic polysaccharide monooxygenase (LPMO), a family 5 chitin-binding domain (CBM5), and a family 18 chitinase (Chi18) domain. The full-length enzyme, which seems to be the only chitinase produced by J. denitrificans, degraded both α- and β-chitin. Both the chitinase and the LPMO activities of Jd1381 were similar to those of other individual chitinases and LPMOs, and the overall efficiency of chitin degradation by full-length Jd1381 depended on its chitinase and LPMO activities. Of note, the chitin-degrading activity of Jd1381 was comparable with or exceeded the activities of combinations of well-known chitinases and an LPMO from Serratia marcescens. Importantly, comparison of the chitinolytic efficiency of Jd1381 with the efficiencies of combinations of truncated variants—JdLPMO10 and JdCBM5-Chi18 or JdLPMO10-CBM5 and JdChi18—indicated that optimal Jd1381 activity requires close spatial proximity of the LPMO10 and the Chi18 domains. The demonstration of intramolecular synergy between LPMOs and hydrolytic enzymes reported here opens new avenues toward the development of efficient catalysts for biomass conversion.

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