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22 pages, 4079 KiB

Open AccessArticle

A Pipeline towards the Biochemical Characterization of the Arabidopsis GT14 Family

by Lingling Xuan, Jie Zhang, Weitai Lu, Pawel Gluza, Berit Ebert, Toshihisa Kotake, Mengzhu Lu, Yuan Zhang, Mads H. Clausen, Kim L. Johnson, Monika S. Doblin, Joshua L. Heazlewood, Antony Bacic, Lili Song and Wei Zeng

Int. J. Mol. Sci. 2021, 22(3), 1360; https://doi.org/10.3390/ijms22031360 - 29 Jan 2021

Cited by 10 | Viewed by 4226

Abstract

Glycosyltransferases (GTs) catalyze the synthesis of glycosidic linkages and are essential in the biosynthesis of glycans, glycoconjugates (glycolipids and glycoproteins), and glycosides. Plant genomes generally encode many more GTs than animal genomes due to the synthesis of a cell wall and a wide [...] Read more.

Glycosyltransferases (GTs) catalyze the synthesis of glycosidic linkages and are essential in the biosynthesis of glycans, glycoconjugates (glycolipids and glycoproteins), and glycosides. Plant genomes generally encode many more GTs than animal genomes due to the synthesis of a cell wall and a wide variety of glycosylated secondary metabolites. The Arabidopsis thaliana genome is predicted to encode over 573 GTs that are currently classified into 42 diverse families. The biochemical functions of most of these GTs are still unknown. In this study, we updated the JBEI Arabidopsis GT clone collection by cloning an additional 105 GT cDNAs, 508 in total (89%), into Gateway-compatible vectors for downstream characterization. We further established a functional analysis pipeline using transient expression in tobacco (Nicotiana benthamiana) followed by enzymatic assays, fractionation of enzymatic products by reversed-phase HPLC (RP-HPLC) and characterization by mass spectrometry (MS). Using the GT14 family as an exemplar, we outline a strategy for identifying effective substrates of GT enzymes. By addition of UDP-GlcA as donor and the synthetic acceptors galactose-nitrobenzodiazole (Gal-NBD), β-1,6-galactotetraose (β-1,6-Gal₄) and β-1,3-galactopentose (β-1,3-Gal₅) to microsomes expressing individual GT14 enzymes, we verified the β-glucuronosyltransferase (GlcAT) activity of three members of this family (AtGlcAT14A, B, and E). In addition, a new family member (AT4G27480, 248) was shown to possess significantly higher activity than other GT14 enzymes. Our data indicate a likely role in arabinogalactan-protein (AGP) biosynthesis for these GT14 members. Together, the updated Arabidopsis GT clone collection and the biochemical analysis pipeline present an efficient means to identify and characterize novel GT catalytic activities. Full article

(This article belongs to the Special Issue Carbohydrate-Active Enzymes: Structure, Activity and Reaction Products 2020)

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23 pages, 3574 KiB

Open AccessArticle

LPMO AfAA9_B and Cellobiohydrolase AfCel6A from A. fumigatus Boost Enzymatic Saccharification Activity of Cellulase Cocktail

by Aline Vianna Bernardi, Luis Eduardo Gerolamo, Paula Fagundes de Gouvêa, Deborah Kimie Yonamine, Lucas Matheus Soares Pereira, Arthur Henrique Cavalcante de Oliveira, Sérgio Akira Uyemura and Taisa Magnani Dinamarco

Int. J. Mol. Sci. 2021, 22(1), 276; https://doi.org/10.3390/ijms22010276 - 29 Dec 2020

Cited by 29 | Viewed by 4696

Abstract

Cellulose is the most abundant polysaccharide in lignocellulosic biomass, where it is interlinked with lignin and hemicellulose. Bioethanol can be produced from biomass. Since breaking down biomass is difficult, cellulose-active enzymes secreted by filamentous fungi play an important role in degrading recalcitrant lignocellulosic [...] Read more.

Cellulose is the most abundant polysaccharide in lignocellulosic biomass, where it is interlinked with lignin and hemicellulose. Bioethanol can be produced from biomass. Since breaking down biomass is difficult, cellulose-active enzymes secreted by filamentous fungi play an important role in degrading recalcitrant lignocellulosic biomass. We characterized a cellobiohydrolase (AfCel6A) and lytic polysaccharide monooxygenase LPMO (AfAA9_B) from Aspergillus fumigatus after they were expressed in Pichia pastoris and purified. The biochemical parameters suggested that the enzymes were stable; the optimal temperature was ~60 °C. Further characterization revealed high turnover numbers (k_cat of 147.9 s⁻¹ and 0.64 s⁻¹, respectively). Surprisingly, when combined, AfCel6A and AfAA9_B did not act synergistically. AfCel6A and AfAA9_B association inhibited AfCel6A activity, an outcome that needs to be further investigated. However, AfCel6A or AfAA9_B addition boosted the enzymatic saccharification activity of a cellulase cocktail and the activity of cellulase Af-EGL7. Enzymatic cocktail supplementation with AfCel6A or AfAA9_B boosted the yield of fermentable sugars from complex substrates, especially sugarcane exploded bagasse, by up to 95%. The synergism between the cellulase cocktail and AfAA9_B was enzyme- and substrate-specific, which suggests a specific enzymatic cocktail for each biomass by up to 95%. The synergism between the cellulase cocktail and AfAA9_B was enzyme- and substrate-specific, which suggests a specific enzymatic cocktail for each biomass. Full article

(This article belongs to the Special Issue Carbohydrate-Active Enzymes: Structure, Activity and Reaction Products 2020)

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16 pages, 2447 KiB

Open AccessArticle

Enzyme Properties of a Laccase Obtained from the Transcriptome of the Marine-Derived Fungus Stemphylium lucomagnoense

by Wissal Ben Ali, Amal Ben Ayed, Annick Turbé-Doan, Emmanuel Bertrand, Yann Mathieu, Craig B. Faulds, Anne Lomascolo, Giuliano Sciara, Eric Record and Tahar Mechichi

Int. J. Mol. Sci. 2020, 21(21), 8402; https://doi.org/10.3390/ijms21218402 - 9 Nov 2020

Cited by 6 | Viewed by 3391

Abstract

Only a few studies have examined how marine-derived fungi and their enzymes adapt to salinity and plant biomass degradation. This work concerns the production and characterisation of an oxidative enzyme identified from the transcriptome of marine-derived fungus Stemphylium lucomagnoense. The laccase-encoding gene [...] Read more.

Only a few studies have examined how marine-derived fungi and their enzymes adapt to salinity and plant biomass degradation. This work concerns the production and characterisation of an oxidative enzyme identified from the transcriptome of marine-derived fungus Stemphylium lucomagnoense. The laccase-encoding gene SlLac2 from S. lucomagnoense was cloned for heterologous expression in Aspergillus niger D15#26 for protein production in the extracellular medium of around 30 mg L⁻¹. The extracellular recombinant enzyme SlLac2 was successfully produced and purified in three steps protocol: ultrafiltration, anion-exchange chromatography, and size exclusion chromatography, with a final recovery yield of 24%. SlLac2 was characterised by physicochemical properties, kinetic parameters, and ability to oxidise diverse phenolic substrates. We also studied its activity in the presence and absence of sea salt. The molecular mass of SlLac2 was about 75 kDa, consistent with that of most ascomycete fungal laccases. With syringaldazine as substrate, SlLac2 showed an optimal activity at pH 6 and retained nearly 100% of its activity when incubated at 50°C for 180 min. SlLac2 exhibited more than 50% of its activity with 5% wt/vol of sea salt. Full article

(This article belongs to the Special Issue Carbohydrate-Active Enzymes: Structure, Activity and Reaction Products 2020)

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22 pages, 5388 KiB

Open AccessArticle

UDP-GLYCOSYLTRANSFERASE 72E3 Plays a Role in Lignification of Secondary Cell Walls in Arabidopsis

by Fabien Baldacci-Cresp, Julien Le Roy, Brigitte Huss, Cédric Lion, Anne Créach, Corentin Spriet, Ludovic Duponchel, Christophe Biot, Marie Baucher, Simon Hawkins and Godfrey Neutelings

Int. J. Mol. Sci. 2020, 21(17), 6094; https://doi.org/10.3390/ijms21176094 - 24 Aug 2020

Cited by 17 | Viewed by 4088

Abstract

Lignin is present in plant secondary cell walls and is among the most abundant biological polymers on Earth. In this work we investigated the potential role of the UGT72E gene family in regulating lignification in Arabidopsis. Chemical determination of floral stem lignin [...] Read more.

Lignin is present in plant secondary cell walls and is among the most abundant biological polymers on Earth. In this work we investigated the potential role of the UGT72E gene family in regulating lignification in Arabidopsis. Chemical determination of floral stem lignin contents in ugt72e1, ugt72e2, and ugt72e3 mutants revealed no significant differences compared to WT plants. In contrast, the use of a novel safranin O ratiometric imaging technique indicated a significant increase in the cell wall lignin content of both interfascicular fibers and xylem from young regions of ugt72e3 mutant floral stems. These results were globally confirmed in interfascicular fibers by Raman microspectroscopy. Subsequent investigation using a bioorthogonal triple labelling strategy suggested that the augmentation in lignification was associated with an increased capacity of mutant cell walls to incorporate H-, G-, and S-monolignol reporters. Expression analysis showed that this increase was associated with an up-regulation of LAC17 and PRX71, which play a key role in lignin polymerization. Altogether, these results suggest that UGT72E3 can influence the kinetics of lignin deposition by regulating monolignol flow to the cell wall as well as the potential of this compartment to incorporate monomers into the growing lignin polymer. Full article

(This article belongs to the Special Issue Carbohydrate-Active Enzymes: Structure, Activity and Reaction Products 2020)

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18 pages, 2727 KiB

Open AccessArticle

Differential Labeling of Glycoproteins with Alkynyl Fucose Analogs

by Chenyu Ma, Hideyuki Takeuchi, Huilin Hao, Chizuko Yonekawa, Kazuki Nakajima, Masamichi Nagae, Tetsuya Okajima, Robert S. Haltiwanger and Yasuhiko Kizuka

Int. J. Mol. Sci. 2020, 21(17), 6007; https://doi.org/10.3390/ijms21176007 - 20 Aug 2020

Cited by 12 | Viewed by 4535

Abstract

Fucosylated glycans critically regulate the physiological functions of proteins and cells. Alterations in levels of fucosylated glycans are associated with various diseases. For detection and functional modulation of fucosylated glycans, chemical biology approaches using fucose (Fuc) analogs are useful. However, little is known [...] Read more.

Fucosylated glycans critically regulate the physiological functions of proteins and cells. Alterations in levels of fucosylated glycans are associated with various diseases. For detection and functional modulation of fucosylated glycans, chemical biology approaches using fucose (Fuc) analogs are useful. However, little is known about how efficiently each unnatural Fuc analog is utilized by enzymes in the biosynthetic pathway of fucosylated glycans. We show here that three clickable Fuc analogs with similar but distinct structures labeled cellular glycans with different efficiency and protein specificity. For instance, 6-alkynyl (Alk)-Fuc modified O-Fuc glycans much more efficiently than 7-Alk-Fuc. The level of GDP-6-Alk-Fuc produced in cells was also higher than that of GDP-7-Alk-Fuc. Comprehensive in vitro fucosyltransferase assays revealed that 7-Alk-Fuc is commonly tolerated by most fucosyltransferases. Surprisingly, both protein O-fucosyltransferases (POFUTs) could transfer all Fuc analogs in vitro, likely because POFUT structures have a larger space around their Fuc binding sites. These findings demonstrate that labeling and detection of fucosylated glycans with Fuc analogs depend on multiple cellular steps, including conversion to GDP form, transport into the ER or Golgi, and utilization by each fucosyltransferase, providing insights into design of novel sugar analogs for specific detection of target glycans or inhibition of their functions. Full article

(This article belongs to the Special Issue Carbohydrate-Active Enzymes: Structure, Activity and Reaction Products 2020)

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16 pages, 4741 KiB

Open AccessArticle

An Exo-Polygalacturonase Pgc4 Regulates Aerial Hyphal Growth and Virulence in Fusarium oxysporum f. sp. cubense race 4

by Zhangyong Dong, Mei Luo and Zhenzhong Wang

Int. J. Mol. Sci. 2020, 21(16), 5886; https://doi.org/10.3390/ijms21165886 - 16 Aug 2020

Cited by 12 | Viewed by 2898

Abstract

Fusarium oxysporum f. sp. cubense race 4 (Foc4) causes Fusarium wilt that affects banana plants, and hence, the molecular mechanisms of its virulence need to be investigated. We purified an exo-polygalacturonase (exo-PG), Pgc4, from Foc4. Pgc4 has an apparent molecular weight of 50.87 [...] Read more.

Fusarium oxysporum f. sp. cubense race 4 (Foc4) causes Fusarium wilt that affects banana plants, and hence, the molecular mechanisms of its virulence need to be investigated. We purified an exo-polygalacturonase (exo-PG), Pgc4, from Foc4. Pgc4 has an apparent molecular weight of 50.87 kDa based on sodium dodecyl sulphate–polyacrylamide gel electrophoresis. We further performed its sequence analysis and biochemical characterization. The two pgc4 genes encoding Pgc4 from Foc4 and Foc1 were 1434 bp in length and encoded 477 amino acids with differences, due to some nucleotide differences between the two. The Km and Vmax values of Pgc4 purified from Foc4 were determined to be 0.45 mg/mL and 105.26 Units·mg·protein⁻¹ ·min⁻¹, respectively. The recombinant proteins, r-Foc1-Pgc4 and r-Foc4-Pgc4, were expressed and purified from Pichia pastoris and showed optimal Pgc4 activity at 55 °C and pH 4.0; both could induce tissue maceration and necrosis in the “Guangfen-1” and “Baxi” varieties of banana but to a different extent. Phenotypic assays and complementation analyses revealed that, compared to the wild-type, the generated Foc4Δpgc4 mutant strain showed a lower aerial hyphal growth, grew slower, and had a reduced virulence. Therefore, our results demonstrate the function of Pgc4 as a pathogenicity factor of Foc4. Full article

(This article belongs to the Special Issue Carbohydrate-Active Enzymes: Structure, Activity and Reaction Products 2020)

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21 pages, 3635 KiB

Open AccessArticle

Dual Substrate Specificity of the Rutinosidase from Aspergillus niger and the Role of Its Substrate Tunnel

by Katerina Brodsky, Michal Kutý, Helena Pelantová, Josef Cvačka, Martin Rebroš, Michael Kotik, Ivana Kutá Smatanová, Vladimír Křen and Pavla Bojarová

Int. J. Mol. Sci. 2020, 21(16), 5671; https://doi.org/10.3390/ijms21165671 - 7 Aug 2020

Cited by 12 | Viewed by 3423

Abstract

Rutinosidases (α-l-rhamnopyranosyl-(1-6)-β-d-glucopyranosidases, EC 3.2.1.168, CAZy GH5) are diglycosidases that cleave the glycosidic bond between the disaccharide rutinose and the respective aglycone. Similar to many retaining glycosidases, rutinosidases can also transfer the rutinosyl moiety onto acceptors with a free –OH [...] Read more.

Rutinosidases (α-l-rhamnopyranosyl-(1-6)-β-d-glucopyranosidases, EC 3.2.1.168, CAZy GH5) are diglycosidases that cleave the glycosidic bond between the disaccharide rutinose and the respective aglycone. Similar to many retaining glycosidases, rutinosidases can also transfer the rutinosyl moiety onto acceptors with a free –OH group (so-called transglycosylation). The recombinant rutinosidase from Aspergillus niger (AnRut) is selectively produced in Pichia pastoris. It can catalyze transglycosylation reactions as an unpurified preparation directly from cultivation. This enzyme exhibits catalytic activity towards two substrates; in addition to rutinosidase activity, it also exhibits β-d-glucopyranosidase activity. As a result, new compounds are formed by β-glucosylation or rutinosylation of acceptors such as alcohols or strong inorganic nucleophiles (NaN₃). Transglycosylation products with aliphatic aglycones are resistant towards cleavage by rutinosidase, therefore, their side hydrolysis does not occur, allowing higher transglycosylation yields. Fourteen compounds were synthesized by glucosylation or rutinosylation of selected acceptors. The products were isolated and structurally characterized. Interactions between the transglycosylation products and the recombinant AnRut were analyzed by molecular modeling. We revealed the role of a substrate tunnel in the structure of AnRut, which explained the unusual catalytic properties of this glycosidase and its specific transglycosylation potential. AnRut is attractive for biosynthetic applications, especially for the use of inexpensive substrates (rutin and isoquercitrin). Full article

(This article belongs to the Special Issue Carbohydrate-Active Enzymes: Structure, Activity and Reaction Products 2020)

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19 pages, 4711 KiB

Open AccessArticle

Mapping the Transglycosylation Relevant Sites of Cold-Adapted β-d-Galactosidase from Arthrobacter sp. 32cB

by Maria Rutkiewicz, Marta Wanarska and Anna Bujacz

Int. J. Mol. Sci. 2020, 21(15), 5354; https://doi.org/10.3390/ijms21155354 - 28 Jul 2020

Cited by 8 | Viewed by 2730

Abstract

β-Galactosidase from Arthrobacter sp. 32cB (ArthβDG) is a cold-adapted enzyme able to catalyze hydrolysis of β-d-galactosides and transglycosylation reaction, where galactosyl moiety is being transferred onto an acceptor larger than a water molecule. Mutants of ArthβDG: D207A and [...] Read more.

β-Galactosidase from Arthrobacter sp. 32cB (ArthβDG) is a cold-adapted enzyme able to catalyze hydrolysis of β-d-galactosides and transglycosylation reaction, where galactosyl moiety is being transferred onto an acceptor larger than a water molecule. Mutants of ArthβDG: D207A and E517Q were designed to determine the significance of specific residues and to enable formation of complexes with lactulose and sucrose and to shed light onto the structural basis of the transglycosylation reaction. The catalytic assays proved loss of function mutation E517 into glutamine and a significant drop of activity for mutation of D207 into alanine. Solving crystal structures of two new mutants, and new complex structures of previously presented mutant E441Q enables description of introduced changes within active site of enzyme and determining the importance of mutated residues for active site size and character. Furthermore, usage of mutants with diminished and abolished enzymatic activity enabled solving six complex structures with galactose, lactulose or sucrose bounds. As a result, not only the galactose binding sites were mapped on the enzyme’s surface but also the mode of lactulose, product of transglycosylation reaction, and binding within the enzyme’s active site were determined and the glucopyranose binding site in the distal of active site was discovered. The latter two especially show structural details of transglycosylation, providing valuable information that may be used for engineering of ArthβDG or other analogous galactosidases belonging to GH2 family. Full article

(This article belongs to the Special Issue Carbohydrate-Active Enzymes: Structure, Activity and Reaction Products 2020)

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24 pages, 14592 KiB

Open AccessArticle

Characterization of the UDP-glycosyltransferase UGT72 Family in Poplar and Identification of Genes Involved in the Glycosylation of Monolignols

by Nathanael Speeckaert, Nassirou Mahamadou Adamou, Hadjara Amadou Hassane, Fabien Baldacci-Cresp, Adeline Mol, Geert Goeminne, Wout Boerjan, Pierre Duez, Simon Hawkins, Godfrey Neutelings, Thomas Hoffmann, Wilfried Schwab, Mondher El Jaziri, Marc Behr and Marie Baucher

Int. J. Mol. Sci. 2020, 21(14), 5018; https://doi.org/10.3390/ijms21145018 - 16 Jul 2020

Cited by 32 | Viewed by 4302

Abstract

Monolignols are the building blocks for lignin polymerization in the apoplastic domain. Monolignol biosynthesis, transport, storage, glycosylation, and deglycosylation are the main biological processes partaking in their homeostasis. In Arabidopsis thaliana, members of the uridine diphosphate-dependent glucosyltransferases UGT72E and UGT72B subfamilies have [...] Read more.

Monolignols are the building blocks for lignin polymerization in the apoplastic domain. Monolignol biosynthesis, transport, storage, glycosylation, and deglycosylation are the main biological processes partaking in their homeostasis. In Arabidopsis thaliana, members of the uridine diphosphate-dependent glucosyltransferases UGT72E and UGT72B subfamilies have been demonstrated to glycosylate monolignols. Here, the poplar UGT72 family, which is clustered into four groups, was characterized: Group 1 UGT72AZ1 and UGT72AZ2, homologs of Arabidopsis UGT72E1-3, as well as group 4 UGT72B37 and UGT72B39, homologs of Arabidopsis UGT72B1-3, glycosylate monolignols. In addition, promoter-GUS analyses indicated that poplar UGT72 members are expressed within vascular tissues. At the subcellular level, poplar UGT72s belonging to group 1 and group 4 were found to be associated with the nucleus and the endoplasmic reticulum. However, UGT72A2, belonging to group 2, was localized in bodies associated with chloroplasts, as well as possibly in chloroplasts. These results show a partial conservation of substrate recognition between Arabidopsis and poplar homologs, as well as divergent functions between different groups of the UGT72 family, for which the substrates remain unknown. Full article

(This article belongs to the Special Issue Carbohydrate-Active Enzymes: Structure, Activity and Reaction Products 2020)

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18 pages, 2718 KiB

Open AccessArticle

Characterisation of the Effect of the Spatial Organisation of Hemicellulases on the Hydrolysis of Plant Biomass Polymer

by Thomas Enjalbert, Marion De La Mare, Pierre Roblin, Louise Badruna, Thierry Vernet, Claire Dumon and Cédric Y. Montanier

Int. J. Mol. Sci. 2020, 21(12), 4360; https://doi.org/10.3390/ijms21124360 - 19 Jun 2020

Cited by 6 | Viewed by 2808

Abstract

Synergism between enzymes is of crucial importance in cell metabolism. This synergism occurs often through a spatial organisation favouring proximity and substrate channelling. In this context, we developed a strategy for evaluating the impact of the geometry between two enzymes involved in nature [...] Read more.

Synergism between enzymes is of crucial importance in cell metabolism. This synergism occurs often through a spatial organisation favouring proximity and substrate channelling. In this context, we developed a strategy for evaluating the impact of the geometry between two enzymes involved in nature in the recycling of the carbon derived from plant cell wall polymers. By using an innovative covalent association process using two protein fragments, Jo and In, we produced two bi-modular chimeric complexes connecting a xylanase and a xylosidase, involved in the deconstruction of xylose-based plant cell wall polymer. We first show that the intrinsic activity of the individual enzymes was preserved. Small Angle X-rays Scattering (SAXS) analysis of the complexes highlighted two different spatial organisations in solution, affecting both the distance between the enzymes (53 Å and 28 Å) and the distance between the catalytic pockets (94 Å and 75 Å). Reducing sugar and HPAEC-PAD analysis revealed different behaviour regarding the hydrolysis of Beechwood xylan. After 24 h of hydrolysis, one complex was able to release a higher amount of reducing sugar compare to the free enzymes (i.e., 15,640 and 14,549 µM of equivalent xylose, respectively). However, more interestingly, the two complexes were able to release variable percentages of xylooligosaccharides compared to the free enzymes. The structure of the complexes revealed some putative steric hindrance, which impacted both enzymatic efficiency and the product profile. This report shows that controlling the spatial geometry between two enzymes would help to better investigate synergism effect within complex multi-enzymatic machinery and control the final product. Full article

(This article belongs to the Special Issue Carbohydrate-Active Enzymes: Structure, Activity and Reaction Products 2020)

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23 pages, 3464 KiB

Open AccessArticle

In-Silico Characterization of Glycosyl Hydrolase Family 1 β-Glucosidase from Trichoderma asperellum UPM1

by Mohamad Farhan Mohamad Sobri, Suraini Abd-Aziz, Farah Diba Abu Bakar and Norhayati Ramli

Int. J. Mol. Sci. 2020, 21(11), 4035; https://doi.org/10.3390/ijms21114035 - 4 Jun 2020

Cited by 8 | Viewed by 3422

Abstract

β-glucosidases (Bgl) are widely utilized for releasing non-reducing terminal glucosyl residues. Nevertheless, feedback inhibition by glucose end product has limited its application. A noticeable exception has been found for β-glucosidases of the glycoside hydrolase (GH) family 1, which exhibit tolerance and even stimulation [...] Read more.

β-glucosidases (Bgl) are widely utilized for releasing non-reducing terminal glucosyl residues. Nevertheless, feedback inhibition by glucose end product has limited its application. A noticeable exception has been found for β-glucosidases of the glycoside hydrolase (GH) family 1, which exhibit tolerance and even stimulation by glucose. In this study, using local isolate Trichoderma asperellum UPM1, the gene encoding β-glucosidase from GH family 1, hereafter designated as TaBgl2, was isolated and characterized via in-silico analyses. A comparison of enzyme activity was subsequently made by heterologous expression in Escherichia coli BL21(DE3). The presence of N-terminal signature, cis-peptide bonds, conserved active site motifs, non-proline cis peptide bonds, substrate binding, and a lone conserved stabilizing tryptophan (W) residue confirms the identity of Trichoderma sp. GH family 1 β-glucosidase isolated. Glucose tolerance was suggested by the presence of 14 of 22 known consensus residues, along with corresponding residues L167 and P172, crucial in the retention of the active site’s narrow cavity. Retention of 40% of relative hydrolytic activity on ρ-nitrophenyl-β-D-glucopyranoside (ρNPG) in a concentration of 0.2 M glucose was comparable to that of GH family 1 β-glucosidase (Cel1A) from Trichoderma reesei. This research thus underlines the potential in the prediction of enzymatic function, and of industrial importance, glucose tolerance of family 1 β-glucosidases following relevant in-silico analyses. Full article

(This article belongs to the Special Issue Carbohydrate-Active Enzymes: Structure, Activity and Reaction Products 2020)

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16 pages, 2293 KiB

Open AccessArticle

Combined Optimization of Codon Usage and Glycine Supplementation Enhances the Extracellular Production of a β-Cyclodextrin Glycosyltransferase from Bacillus sp. NR5 UPM in Escherichia coli

by Nik Ida Mardiana Nik-Pa, Mohamad Farhan Mohamad Sobri, Suraini Abd-Aziz, Mohamad Faizal Ibrahim, Ezyana Kamal Bahrin, Noorjahan Banu Mohammed Alitheen and Norhayati Ramli

Int. J. Mol. Sci. 2020, 21(11), 3919; https://doi.org/10.3390/ijms21113919 - 30 May 2020

Cited by 15 | Viewed by 3382

Abstract

Two optimization strategies, codon usage modification and glycine supplementation, were adopted to improve the extracellular production of Bacillus sp. NR5 UPM β-cyclodextrin glycosyltransferase (CGT-BS) in recombinant Escherichia coli. Several rare codons were eliminated and replaced with the ones favored by E. coli [...] Read more.

Two optimization strategies, codon usage modification and glycine supplementation, were adopted to improve the extracellular production of Bacillus sp. NR5 UPM β-cyclodextrin glycosyltransferase (CGT-BS) in recombinant Escherichia coli. Several rare codons were eliminated and replaced with the ones favored by E. coli cells, resulting in an increased codon adaptation index (CAI) from 0.67 to 0.78. The cultivation of the codon modified recombinant E. coli following optimization of glycine supplementation enhanced the secretion of β-CGTase activity up to 2.2-fold at 12 h of cultivation as compared to the control. β-CGTase secreted into the culture medium by the transformant reached 65.524 U/mL at post-induction temperature of 37 °C with addition of 1.2 mM glycine and induced at 2 h of cultivation. A 20.1-fold purity of the recombinant β-CGTase was obtained when purified through a combination of diafiltration and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. This combined strategy doubled the extracellular β-CGTase production when compared to the single approach, hence offering the potential of enhancing the expression of extracellular enzymes, particularly β-CGTase by the recombinant E. coli. Full article

(This article belongs to the Special Issue Carbohydrate-Active Enzymes: Structure, Activity and Reaction Products 2020)

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13 pages, 3527 KiB

Open AccessArticle

The Crystal Structure of a Streptomyces thermoviolaceus Thermophilic Chitinase Known for Its Refolding Efficiency

by Piotr H. Malecki, Magdalena Bejger, Wojciech Rypniewski and Constantinos E. Vorgias

Int. J. Mol. Sci. 2020, 21(8), 2892; https://doi.org/10.3390/ijms21082892 - 21 Apr 2020

Cited by 11 | Viewed by 3370

Abstract

Analyzing the structure of proteins from extremophiles is a promising way to study the rules governing the protein structure, because such proteins are results of structural and functional optimization under well-defined conditions. Studying the structure of chitinases addresses an interesting aspect of enzymology, [...] Read more.

Analyzing the structure of proteins from extremophiles is a promising way to study the rules governing the protein structure, because such proteins are results of structural and functional optimization under well-defined conditions. Studying the structure of chitinases addresses an interesting aspect of enzymology, because chitin, while being the world’s second most abundant biopolymer, is also a recalcitrant substrate. The crystal structure of a thermostable chitinase from Streptomyces thermoviolaceus (StChi40) has been solved revealing a β/α-barrel (TIM-barrel) fold with an α+β insertion domain. This is the first chitinase structure of the multi-chitinase system of S. thermoviolaceus. The protein is also known to refold efficiently after thermal or chemical denaturation. StChi40 is structurally close to the catalytic domain of psychrophilic chitinase B from Arthrobacter TAD20. Differences are noted in comparison to the previously examined chitinases, particularly in the substrate-binding cleft. A comparison of the thermophilic enzyme with its psychrophilic homologue revealed structural features that could be attributed to StChi40’s thermal stability: compactness of the structure with trimmed surface loops and unique disulfide bridges, one of which is additionally stabilized by S–π interactions with aromatic rings. Uncharacteristically for thermophilic proteins, StChi40 has fewer salt bridges than its mesophilic and psychrophilic homologues. Full article

(This article belongs to the Special Issue Carbohydrate-Active Enzymes: Structure, Activity and Reaction Products 2020)

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18 pages, 1740 KiB

Open AccessArticle

Comparative Analysis of High-Throughput Assays of Family-1 Plant Glycosyltransferases

by Kate McGraphery and Wilfried Schwab

Int. J. Mol. Sci. 2020, 21(6), 2208; https://doi.org/10.3390/ijms21062208 - 23 Mar 2020

Cited by 15 | Viewed by 5807

Abstract

The ability of glycosyltransferases (GTs) to reduce volatility, increase solubility, and thus alter the bioavailability of small molecules through glycosylation has attracted immense attention in pharmaceutical, nutraceutical, and cosmeceutical industries. The lack of GTs known and the scarcity of high-throughput (HTP) available methods, [...] Read more.

The ability of glycosyltransferases (GTs) to reduce volatility, increase solubility, and thus alter the bioavailability of small molecules through glycosylation has attracted immense attention in pharmaceutical, nutraceutical, and cosmeceutical industries. The lack of GTs known and the scarcity of high-throughput (HTP) available methods, hinders the extrapolation of further novel applications. In this study, the applicability of new GT-assays suitable for HTP screening was tested and compared with regard to harmlessness, robustness, cost-effectiveness and reproducibility. The UDP-Glo GT-assay, Phosphate GT Activity assay, pH-sensitive GT-assay, and UDP²-TR-FRET assay were applied and tailored to plant UDP GTs (UGTs). Vitis vinifera (UGT72B27) GT was subjected to glycosylation reaction with various phenolics. Substrate screening and kinetic parameters were evaluated. The pH-sensitive assay and the UDP²-TR-FRET assay were incomparable and unsuitable for HTP plant GT-1 family UGT screening. Furthermore, the UDP-Glo GT-assay and the Phosphate GT Activity assay yielded closely similar and reproducible K_M, v_max, and k_cat values. Therefore, with the easy experimental set-up and rapid readout, the two assays are suitable for HTP screening and quantitative kinetic analysis of plant UGTs. This research sheds light on new and emerging HTP assays, which will allow for analysis of novel family-1 plant GTs and will uncover further applications. Full article

(This article belongs to the Special Issue Carbohydrate-Active Enzymes: Structure, Activity and Reaction Products 2020)

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12 pages, 1865 KiB

Open AccessArticle

Overexpression and Biochemical Characterization of an Endo-α-1,4-polygalacturonase from Aspergillus nidulans in Pichia pastoris

by Hua Xu, Pengfei Zhang, Yuchen Zhang, Zebin Liu, Xuebing Zhang, Zhimin Li, Jian-Jun Li and Yuguang Du

Int. J. Mol. Sci. 2020, 21(6), 2100; https://doi.org/10.3390/ijms21062100 - 19 Mar 2020

Cited by 11 | Viewed by 2797

Abstract

Pectinases have many applications in the industry of food, paper, and textiles, therefore finding novel polygalacturonases is required. Multiple sequence alignment and phylogenetic analysis of AnEPG (an endo-α-1,4-polygalacturonase from Aspergillus nidulans) and other GH 28 endo-polygalacturonases suggested that AnEPG is different from [...] Read more.

Pectinases have many applications in the industry of food, paper, and textiles, therefore finding novel polygalacturonases is required. Multiple sequence alignment and phylogenetic analysis of AnEPG (an endo-α-1,4-polygalacturonase from Aspergillus nidulans) and other GH 28 endo-polygalacturonases suggested that AnEPG is different from others. AnEPG overexpressed in Pichia pastoris was characterized. AnEPG showed the highest activity at pH 4.0, and exhibited moderate activity over a narrow pH range (pH 2.0–5.0) and superior stability in a wide pH range (pH 2.0–12.0). It displayed the highest activity at 60 °C, and retained >42.2% of maximum activity between 20 and 80 °C. It was stable below 40 °C and lost activity very quickly above 50 °C. Its apparent kinetic parameters against PGA (polygalacturonic acid) were determined, with the K_m and k_cat values of 8.3 mg/mL and 5640 μmol/min/mg, respectively. Ba²⁺ and Ni²⁺ enhanced activity by 12.2% and 9.4%, respectively, while Ca²⁺, Cu²⁺, and Mn²⁺ inhibited activity by 14.8%, 12.8%, and 10.2% separately. Analysis of hydrolysis products by AnEPG proved that AnEPG belongs to an endo-polygalacturonase. Modelled structure of AnEPG by I-TASSER showed structural characteristics of endo-polygalacturonases. This pectinase has great potential to be used in food industry and as feed additives. Full article

(This article belongs to the Special Issue Carbohydrate-Active Enzymes: Structure, Activity and Reaction Products 2020)

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Review

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34 pages, 2064 KiB

Open AccessReview

LytR-CpsA-Psr Glycopolymer Transferases: Essential Bricks in Gram-Positive Bacterial Cell Wall Assembly

by Cordula Stefanović, Fiona F. Hager and Christina Schäffer

Int. J. Mol. Sci. 2021, 22(2), 908; https://doi.org/10.3390/ijms22020908 - 18 Jan 2021

Cited by 18 | Viewed by 5102

Abstract

The cell walls of Gram-positive bacteria contain a variety of glycopolymers (CWGPs), a significant proportion of which are covalently linked to the peptidoglycan (PGN) scaffolding structure. Prominent CWGPs include wall teichoic acids of Staphylococcus aureus, streptococcal capsules, mycobacterial arabinogalactan, and rhamnose-containing polysaccharides [...] Read more.

The cell walls of Gram-positive bacteria contain a variety of glycopolymers (CWGPs), a significant proportion of which are covalently linked to the peptidoglycan (PGN) scaffolding structure. Prominent CWGPs include wall teichoic acids of Staphylococcus aureus, streptococcal capsules, mycobacterial arabinogalactan, and rhamnose-containing polysaccharides of lactic acid bacteria. CWGPs serve important roles in bacterial cellular functions, morphology, and virulence. Despite evident differences in composition, structure and underlaying biosynthesis pathways, the final ligation step of CWGPs to the PGN backbone involves a conserved class of enzymes—the LytR-CpsA-Psr (LCP) transferases. Typically, the enzymes are present in multiple copies displaying partly functional redundancy and/or preference for a distinct CWGP type. LCP enzymes require a lipid-phosphate-linked glycan precursor substrate and catalyse, with a certain degree of promiscuity, CWGP transfer to PGN of different maturation stages, according to in vitro evidence. The prototype attachment mode is that to the C6-OH of N-acetylmuramic acid residues via installation of a phosphodiester bond. In some cases, attachment proceeds to N-acetylglucosamine residues of PGN—in the case of the Streptococcus agalactiae capsule, even without involvement of a phosphate bond. A novel aspect of LCP enzymes concerns a predicted role in protein glycosylation in Actinomyces oris. Available crystal structures provide further insight into the catalytic mechanism of this biologically important class of enzymes, which are gaining attention as new targets for antibacterial drug discovery to counteract the emergence of multidrug resistant bacteria. Full article

(This article belongs to the Special Issue Carbohydrate-Active Enzymes: Structure, Activity and Reaction Products 2020)

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16 pages, 2693 KiB

Open AccessReview

Galactofuranose-Related Enzymes: Challenges and Hopes

by Mateja Seničar, Pierre Lafite, Svetlana V. Eliseeva, Stéphane Petoud, Ludovic Landemarre and Richard Daniellou

Int. J. Mol. Sci. 2020, 21(10), 3465; https://doi.org/10.3390/ijms21103465 - 14 May 2020

Cited by 10 | Viewed by 4933

Abstract

Galactofuranose is a rare form of the well-known galactose sugar, and its occurrence in numerous pathogenic micro-organisms makes the enzymes responsible for its biosynthesis interesting targets. Herein, we review the role of these carbohydrate-related proteins with a special emphasis on the galactofuranosidases we [...] Read more.

Galactofuranose is a rare form of the well-known galactose sugar, and its occurrence in numerous pathogenic micro-organisms makes the enzymes responsible for its biosynthesis interesting targets. Herein, we review the role of these carbohydrate-related proteins with a special emphasis on the galactofuranosidases we recently characterized as an efficient recombinant biocatalyst. Full article

(This article belongs to the Special Issue Carbohydrate-Active Enzymes: Structure, Activity and Reaction Products 2020)

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Graphical abstract

19 pages, 1623 KiB

Open AccessReview

Sucrose Phosphorylase and Related Enzymes in Glycoside Hydrolase Family 13: Discovery, Application and Engineering

by Jorick Franceus and Tom Desmet

Int. J. Mol. Sci. 2020, 21(7), 2526; https://doi.org/10.3390/ijms21072526 - 5 Apr 2020

Cited by 58 | Viewed by 10616

Abstract

Sucrose phosphorylases are carbohydrate-active enzymes with outstanding potential for the biocatalytic conversion of common table sugar into products with attractive properties. They belong to the glycoside hydrolase family GH13, where they are found in subfamily 18. In bacteria, these enzymes catalyse the phosphorolysis [...] Read more.

Sucrose phosphorylases are carbohydrate-active enzymes with outstanding potential for the biocatalytic conversion of common table sugar into products with attractive properties. They belong to the glycoside hydrolase family GH13, where they are found in subfamily 18. In bacteria, these enzymes catalyse the phosphorolysis of sucrose to yield α-glucose 1-phosphate and fructose. However, sucrose phosphorylases can also be applied as versatile transglucosylases for the synthesis of valuable glycosides and sugars because their broad promiscuity allows them to transfer the glucosyl group of sucrose to a diverse collection of compounds other than phosphate. Numerous process and enzyme engineering studies have expanded the range of possible applications of sucrose phosphorylases ever further. Moreover, it has recently been discovered that family GH13 also contains a few novel phosphorylases that are specialised in the phosphorolysis of sucrose 6^F-phosphate, glucosylglycerol or glucosylglycerate. In this review, we provide an overview of the progress that has been made in our understanding and exploitation of sucrose phosphorylases and related enzymes over the past ten years. Full article

(This article belongs to the Special Issue Carbohydrate-Active Enzymes: Structure, Activity and Reaction Products 2020)

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