GH Family Research Articles

Immobilization of α-transglucosidase on silica-coated magnetic nanoparticles and its application for production of isomaltooligosaccharide from the potato peel

In this study, the production of isomaltooligosaccharide from potato peel starch was carried out in three steps: liquefaction, saccharification, and transglucosylation. Further, cloning α-transglucosidase gene from Aspergillus niger (GH31 family), transforming into E. coli BL21 (DE3), overexpressing and purifying the resulting protein for the production of α-transglucosidase. The generated α-transglucosidase was then bound with magnetic nanoparticles, which improved reusability up to 5 cycles with more than 60% activity. All the modifications were characterized using the following methods: Fourier transform infra-red analysis, Transmission Electron Microscopy, Field Emission Scanning Electron Microscopy, Energy Dispersive X-ray spectroscopy, X-Ray Diffraction Spectroscopy, Thermogravimetric Analysis, and Dynamic Light Scattering (DLS) analysis. Further, the optimum conditions for transglucosylation were determined by RSM as follows: enzyme-to-substrate ratio 6.9 U g−1, reaction time 9 h, temperature 45 °C, and pH 5.5 with a yield of 70 g l−1 (± 2.1). MALDI-TOF–MS analysis showed DP of the IMOs in ranges of 2–10. The detailed structural characterization of isomaltooligosaccharide by GC–MS and NMR suggested the α-(1 → 4) and α-(1 → 6)-D-Glcp residues as major constituents along with minor α-(1 → 2) and α-(1 → 3) -D-Glcp residues.

Cloning and biochemical characterization of a recombinant chitinase encoded by the CHT4 gene from Candida albicans

As one of the major components in the fungal cell wall, chitin is a polymer of β-1,4-linked N-acetylglucosamine. Chitinases are hydrolytic enzymes that break down glycosidic bonds in the chitin. The human fungal pathogen Candida albicans has three chitinase-encoding genes, CaCHT1, CaCHT2 and CaCHT3. The CaCHT4 gene encodes a protein with the glycoside hydrolase family GH18 domain, Glyco_18, which suggests that CaCht4 might be a chitinase. In the present study, we have cloned, expressed and purified the N-terminally His6-tagged CaCht4 protein from bacterial cells. Further biochemical characterization has shown that this recombinant CaCht4 protein shows both exochitinase (chitobiosidase) and endochitinase activities, but has no N-acetylglucosaminase activity. The optimal temperature for the exochitinase activity of CaCht4 is 55 °C. Taken together, these data support that the CaCHT4 gene encodes a chitinase. Our finding provides a basis for us to understand the biological functions of the CaCHT4 gene in C. albicans.

Propeptide-Mediated Allosteric Regulation of Xylanase Xyl-1: An Integrated Experimental and Computational Analysis.

Most GH11 family endo-β-1,4-xylanases contain a propeptide region linked to the N-terminal region. The mechanistic basis of this region harboring key regulation information for enzyme function, however, remains poorly understood. We reported an investigation on the allosteric regulation mechanism of the propeptide based on biochemical characterization, molecular dynamics simulations, and evolutionary analysis. We discovered that the mutant of truncated propeptide shows a remarkably increased thermal stability (melting temperature increased by 11.5 °C) and catalytic efficiency (1.7-fold kcat/Km value of wild type). Molecular dynamics simulations reveal that long-range fluctuations in the propeptide lead to a conformational perturbation in the catalytic pocket and the thumb region. The probability of sampling the active conformation during the glycosylation step is reduced (i.e., catalytic efficiency). In-depth sequence analysis indicates that the propeptide has a strong plasticity and degeneration trend, and propeptide truncation experiments of the homologous enzyme XynB validated the feasibility of the truncation strategy. This work reveals the role of GH11 family propeptides in functional regulation and provides a straightforward and practical method to increase the robustness of GH11 family xylanases.

Production and Characterization of Cellulose Nanocrystals from Eucalyptus Dissolving Pulp Using Endoglucanases from Myceliophthora thermophila

Endoglucanase (EG) is a key enzyme during enzymatic preparation of cellulose nanocrystals (CNCs). Myceliophthora thermophila is a thermophilic fungus that has thermal properties and a high secretion of endoglucanases (EGs), and could serve as potential sources of EGs for the preparation of CNCs. In this work, four different GH families (GH5, GH7, GH12, and GH45) of EGs from M. thermophila were expressed and purified, and their enzymatic characteristics and feasibility of application in CNC preparation were investigated. It was shown that the MtEG5A from M. thermophila has good potential in the enzymatic preparation of CNCs using eucalyptus dissolving pulp as feedstock. It was also observed that there was a synergistic effect between the MtEG5A and other MtEGs in the preparation of CNCs, which improved the yield and properties of CNCs obtained by enzymatic hydrolysis. This study provides a reference for understanding the enzymatic characteristics of different families of EGs from M. thermophile and their potential application in nanocellulose production.

Comparative Transcriptome Analysis between Embryogenic and Non-Embryogenic Callus of Davidia involucrata

Davidia involucrata Baill. (D. involucrata), a rare and endangered wild plant, is native to China and is globally recognized as an ornamental tree species. However, D. involucrata exhibits inherent biological characteristics that contribute to its low reproductive efficiency. To address this challenge, somatic embryogenesis, a biotechnological method, offers numerous advantages, including enhanced reproductive efficiency, a large reproductive coefficient, and a complete structural composition. Consequently, somatic embryogenesis holds significant value in the propagation and genetic improvement of this particular tree species. In a previous study, we utilized immature zygotic embryos of D. involucrata as explants and induced somatic embryogenesis from embryogenic callus, thereby establishing a rapid propagation and plant regeneration scheme. In this study, we utilized Illumina RNA sequencing to compare the transcriptomes of the embryogenic callus (EC) and non-embryogenic callus (NEC) of D. involucrata. The analysis revealed 131,109 unigenes assembled from EC and NEC, and 12,806 differentially expressed genes (DEGs) were identified. To verify the authenticity of the transcriptome sequencing results, qRT-PCR was performed and 16 DEGs were screened, with the stable reference gene UBQ being selected. Our analysis focused on genes related to plant growth regulators and somatic embryogenesis, such as the Aux, IAA, ARF, GH3, AHP, ARR, CYCD, BBM, WUS, GRF, SERK, and WOX gene families. We found that certain genes in these families were significantly upregulated in EC induction compared to NEC, indicating that they play crucial roles in D. involucrata cell proliferation, differentiation, and cell totipotency. These results offer new insights into the role of these gene families in EC, and may guide efforts to improve the somatic embryo induction, culture conditions, and genetic transformation efficiency of D. involucrata.

Biochemical characterization and elucidation of the action mode of a GH16 family κ-carrageenase for efficient preparation of carrageenan oligosaccharides.

κ-Carrageenan oligosaccharides have a variety of biological activities. Degradation of κ-carrageenan by κ-carrageenase leads to degradation products with different degrees of polymerization (DPs). A novel gene (CecgkA) encoding a new κ-carrageenase was cloned from Colwellia echini and heterologously expressed in Escherichia coli BL21 (DE3). The enzyme is 1104bp in length, encodes 367 amino acid residues and has a molecular weight of 41.30kDa. Multiple alignment analysis showed that CeCgkA belongs to the glycoside hydrolase (GH16) family and has the highest homology with the κ-carrageenase of Rhodopirellula maiorica SM1, with 58% homology. The CeCgkA showed maximum activity (453.15 U/mg) at pH 8.0 and 35°C. Determination of biochemical properties showed that CeCgkA was a thermal recovery enzyme, and 51.6% of the initial enzyme activity was recovered by immediately placing the sample at 35°C for 60min after enzymatic inactivation by boiling for 10min. K+, Na+, and EDTA had an activating effect on the enzyme activity, while Ni2+, Cu2+, and Zn2+ inhibited the activity of the enzyme. In addition, TLC and ESI-MS analysis showed that the maximum recognition unit of CecgkA was decasaccharide and that the main degradation products were disaccharides, tetrasaccharides and hexasaccharides, indicating that the enzyme is an endo-type carrageenase.

Molecular mechanisms of processive glycoside hydrolases underline catalytic pragmatism.

Processive and distributive catalysis defines the conversion continuum, thus underpinning the transformation of oligo- and polymeric substrates by enzymes. Distributive catalysis follows an association-transformation-dissociation pattern during the formation of enzyme-reactant complexes, whereas during processive catalysis, enzymes partner with substrates and complete multiple catalytic events before dissociation from an enzyme-substrate complex. Here, we focus on processive catalysis in glycoside hydrolases (GHs), which ensures efficient conversions of substrates with high precision, and has the advantage over distributive catalysis in efficiency. The work presented here examines a recent discovery of substrate-product-assisted processive catalysis in the GH3 family enzymes with enclosed pocket-shaped active sites. We detail how GH3 β-d-glucan glucohydrolases exploit a transiently formed lateral pocket for product displacement and reactants sliding (or translocation motion) through the catalytic site without dissociation, including movements during nanoscale binding/unbinding and sliding. The phylogenetic tree of putative 550 Archaean, bacterial, fungal, Viridiplantae, and Metazoan GH3 entries resolved seven lineages that corresponded to major substrate specificity groups. This analysis indicates that two tryptophan residues in plant β-d-glucan glucohydrolases that delineate the catalytic pocket, and infer broad specificity, high catalytic efficiency, and substrate-product-assisted processivity, have evolved through a complex evolutionary process, including horizontal transfer and neo-functionalisation. We conclude that the definition of thermodynamic and mechano-structural properties of processive enzymes is fundamentally important for theoretical and practical applications in bioengineering applicable in various biotechnologies.

Cellulose metabolism in halo(natrono)archaea: a comparative genomics study.

Extremely halophilic archaea are one of the principal microbial community components in hypersaline environments. The majority of cultivated haloarchaea are aerobic heterotrophs using peptides or simple sugars as carbon and energy sources. At the same time, a number of novel metabolic capacities of these extremophiles were discovered recently among which is a capability of growing on insoluble polysaccharides such as cellulose and chitin. Still, polysaccharidolytic strains are in minority among cultivated haloarchaea and their capacities of hydrolyzing recalcitrant polysaccharides are hardly investigated. This includes the mechanisms and enzymes involved in cellulose degradation, which are well studied for bacterial species, while almost unexplored in archaea and haloarchaea in particular. To fill this gap, a comparative genomic analysis of 155 cultivated representatives of halo(natrono)archaea, including seven cellulotrophic strains belonging to the genera Natronobiforma, Natronolimnobius, Natrarchaeobius, Halosimplex, Halomicrobium and Halococcoides was performed. The analysis revealed a number of cellulases, encoded in the genomes of cellulotrophic strains but also in several haloarchaea, for which the capacity to grow on cellulose was not shown. Surprisingly, the cellulases genes, especially of GH5, GH9 and GH12 families, were significantly overrepresented in the cellulotrophic haloarchaea genomes in comparison with other cellulotrophic archaea and even cellulotrophic bacteria. Besides cellulases, the genes for GH10 and GH51 families were also abundant in the genomes of cellulotrophic haloarchaea. These results allowed to propose the genomic patterns, determining the capability of haloarchaea to grow on cellulose. The patterns helped to predict cellulotrophic capacity for several halo(natrono)archaea, and for three of them it was experimentally confirmed. Further genomic search revealed that glucose and cellooligosaccharides import occurred by means of porters and ABC (ATP-binding cassette) transporters. Intracellular glucose oxidation occurred through glycolysis or the semi-phosphorylative Entner-Dudoroff pathway which occurrence was strain-specific. Comparative analysis of CAZymes toolbox and available cultivation-based information allowed proposing two possible strategies used by haloarchaea capable of growing on cellulose: so-called specialists are more effective in degradation of cellulose while generalists are more flexible in nutrient spectra. Besides CAZymes profiles the groups differed in genome sizes, as well as in variability of mechanisms of import and central metabolism of sugars.

Biochemical Properties of a Cold-Active Chitinase from Marine Trichoderma gamsii R1 and Its Application to Preparation of Chitin Oligosaccharides

The enzymatic degradation of different chitin polymers into chitin oligosaccharides (COSs) is of great significance given their better solubility and various biological applications. Chitinase plays a pivotal role in the enzymatic preparation of COSs. Herein, a cold-adapted and efficient chitinase (ChiTg) from the marine Trichoderma gamsii R1 was purified and characterized. The optimal temperature of ChiTg was 40 °C, and the relative activity at 5 °C was above 40.1%. Meanwhile, ChiTg was active and stable from pH 4.0 to 7.0. As an endo-type chitinase, ChiTg exhibited the highest activity with colloidal chitin, then with ball-milled and powdery chitin. In addition, ChiTg showed high efficiency when hydrolyzing colloidal chitin at different temperatures, and the end products were mainly composed of COSs with one to three degrees of polymerization. Furthermore, the results of bioinformatics analysis revealed that ChiTg belongs to the GH18 family, and its acidic surface and the flexible structure of its catalytic site may contribute to its high activity in cold conditions. The results of this study provide a cold-active and efficient chitinase and ideas for its application regarding the preparation of COSs from colloidal chitin.

Molecular dynamics-based structural insights of the first putative endoglucanase, PsGH5A of glycoside hydrolase family 5 from Pseudopedobacter saltans.

The putative endoglucanase, PsGH5A, from Pseudopedobacter saltans of family GH5 contains a catalytic module, PsGH5 (β/α)8-TIM barrel), at N-terminal followed by a family 6 carbohydrate-binding module (CBM6, β-sandwich). Superposition of PsGH5A with PDB homologs revealed Glu220 and Glu318 as evolutionarily conserved and catalytic residues performing the hydrolysis through retaining-type mechanism, a canonical property of GH5 family. PsGH5A showed higher affinity for longer cellooligosaccharides, as long as cellodecaose with binding free energy (∆G) of - 13.72kcal/mol upon the molecular docking, thereby indicating the endo-mode of hydrolysis. The radius of gyration, Rg (2.7nm), and solvent accessible surface area, SASA (229.6 nm2), of PsGH5A-Cellotetraose complex were determined by MD simulation which was lower than that of PsGH5A (Rg, 2.8nm, SASA, 267 nm2) demonstrating the compactness and affinity of PsGH5A with the cellulosic ligands. Cellulose compatibility of PsGH5A was further confirmed by MMPBSA and per-residue decomposition analysis, where notable ∆G of - 54.38kcal/mol for PsGH5A-Cellotetraose complex was observed. Thus, PsGH5A could be potentially an efficient endoglucanase as it accommodated larger cellooligosaccharides at its active-site. PsGH5A is the first putative endoglucanase studied here from P. saltans which could be genome-mined for lignocellulosic biomass saccharification in the renewable energy sector. The 3-D structure of PsGH5A generated by AlphaFold2, RaptorX, SwissModel, Phyre2 and Robetta tool; YASARA was used for energy minimization of built models. UCLA SAVES-v6 was used for quality assessment of models. Molecular Docking was performed using SWISS-DOCK server and Chimera software. Molecular Dynamics simulations and MMPBSA analysis of PsGH5A and PsGH5A-Cellotetraose complex were performed on GROMACS 2019.6.

Chitinolytic Enzymes of the Hyperparasite Fungus Aphanocladium album: Genome-Wide Survey and Characterization of A Selected Enzyme.

The filamentous fungus Aphanocladium album is known as a hyperparasite of plant pathogenic fungi; hence, it has been studied as a possible agent for plant protection. Chitinases secreted by A. album have proven to be essential for its fungicidal activity. However, no complete analysis of the A. album chitinase assortment has been carried out, nor have any of its chitinases been characterized yet. In this study, we report the first draft assembly of the genome sequence of A. album (strain MX-95). The in silico functional annotation of the genome allowed the identification of 46 genes encoding chitinolytic enzymes of the GH18 (26 genes), GH20 (8 genes), GH75 (8 genes), and GH3 (4 genes) families. The encoded proteins were investigated by comparative and phylogenetic analysis, allowing clustering in different subgroups. A. album chitinases were also characterized according to the presence of different functional protein domains (carbohydrate-binding modules and catalytic domains) providing the first complete description of the chitinase repertoire of A. album. A single chitinase gene was then selected for complete functional characterization. The encoded protein was expressed in the yeast Pichia pastoris, and its activity was assayed under different conditions of temperature and pH and with different substrates. It was found that the enzyme acts mainly as a chitobiosidase, with higher activity in the 37-50 °C range.

Functional annotation of rhizospheric phageome of the wild plant species Moringa oleifera.

The study aims to describe phageome of soil rhizosphere of M.oleifera in terms of the genes encoding CAZymes and other KEGG enzymes. Genes of the rhizospheric virome of the wild plant species Moringa oleifera were investigated for their ability to encode useful CAZymes and other KEGG (Kyoto Encyclopedia of Genes and Genomes) enzymes and to resist antibiotic resistance genes (ARGs) in the soil. Abundance of these genes was higher in the rhizospheric microbiome than in the bulk soil. Detected viral families include the plant viral family Potyviridae as well as the tailed bacteriophages of class Caudoviricetes that are mainly associated with bacterial genera Pseudomonas, Streptomyces and Mycobacterium. Viral CAZymes in this soil mainly belong to glycoside hydrolase (GH) families GH43 and GH23. Some of these CAZymes participate in a KEGG pathway with actions included debranching and degradation of hemicellulose. Other actions include biosynthesizing biopolymer of the bacterial cell wall and the layered cell wall structure of peptidoglycan. Other CAZymes promote plant physiological activities such as cell-cell recognition, embryogenesis and programmed cell death (PCD). Enzymes of other pathways help reduce the level of soil H2O2 and participate in the biosynthesis of glycine, malate, isoprenoids, as well as isoprene that protects plant from heat stress. Other enzymes act in promoting both the permeability of bacterial peroxisome membrane and carbon fixation in plants. Some enzymes participate in a balanced supply of dNTPs, successful DNA replication and mismatch repair during bacterial cell division. They also catalyze the release of signal peptides from bacterial membrane prolipoproteins. Phages with the most highly abundant antibiotic resistance genes (ARGs) transduce species of bacterial genera Pseudomonas, Streptomyces, and Mycobacterium. Abundant mechanisms of antibiotic resistance in the rhizosphere include "antibiotic efflux pump" for ARGs soxR, OleC, and MuxB, "antibiotic target alteration" for parY mutant, and "antibiotic inactivation" for arr-1. These ARGs can act synergistically to inhibit several antibiotics including tetracycline, penam, cephalosporin, rifamycins, aminocoumarin, and oleandomycin. The study highlighted the issue of horizontal transfer of ARGs to clinical isolates and human gut microbiome.

α-L-rhamnosidase: production, properties, and applications.

α-L-rhamnosidase [EC 3.2.1.40] belongs to glycoside hydrolase (GH) families (GH13, GH78, and GH106 families) in the carbohydrate-active enzymes (CAZy) database, which specifically hydrolyzes the non-reducing end of α-L-rhamnose. Αccording to the sites of catalytic hydrolysis, α-L-rhamnosidase can be divided into α-1, 2-rhamnosidase, α-1, 3-rhamnosidase, α-1, 4-rhamnosidase and α-1, 6-rhamnosidase. α-L-rhamnosidase is an important enzyme for various biotechnological applications, especially in food, beverage, and pharmaceutical industries. α-L-rhamnosidase has a wide range of sources and is commonly found in animals, plants, and microorganisms, and its microbial source includes a variety of bacteria, molds and yeasts (such as Lactobacillus sp., Aspergillus sp., Pichia angusta and Saccharomyces cerevisiae). In recent years, a series of advances have been achieved in various aspects of α-validates the above-described-rhamnosidase research. A number of α-L-rhamnosidases have been successfully recombinant expressed in prokaryotic systems as well as eukaryotic systems which involve Pichia pastoris, Saccharomyces cerevisiae and Aspergillus niger, and the catalytic properties of the recombinant enzymes have been improved by enzyme modification techniques. In this review, the sources and production methods, general and catalytic properties and biotechnological applications of α-L-rhamnosidase in different fields are summarized and discussed, concluding with the directions for further in-depth research on α-L-rhamnosidase.

Deciphering the structure of a distinctive trimodular cellulosomal licheninase (RfGH16_21), a family 16 glycoside hydrolase from Ruminococcus flavefaciens by computational and experimental methods

In order to know the insights of a unique naturally existing trimodular licheninase from GH16 family, sub-family 21 (RfGH16_21) from Ruminococcus flavefaciens, its structure was modeled to understand its functional relations to reveal information regarding modifying the enzyme for improved properties with enhanced catalytic efficiency. Homology modeling revealed three tandem repeats of β-jelly roll like folds linked by natural linkers. Catalytic pockets and the catalytically important amino acids in each tandem repeat of RfGH16_21 determined by multiple sequence alignment and structure superposition with its homologues indicated that two Glu residues are involved in a retaining-type of catalytic mechanism. Sequential molecular docking revealed maximum binding energy with mixed linked cellotriose showing that cellotriose is the lowest oligomeric hydrolysed product formed by the catalytic action of endo-β-1,3–1,4-glucanase. Molecular dynamic (MD) simulation of RfGH16_21-cellotriose complex confirmed the structural specificity of catalytic residues and increased stability of enzyme in presence of ligand as compared to simulated RfGH16_21 alone. The binding affinity of cellotriose towards the three tandem repeats of RfGH16_21 was also confirmed by calculating total binding Gibbs free energy, i.e. −100.8 ± 2.6 KJ/mol, by using g_mmpbsa tool. The stability of the protein was determined by protein melting analysis that showed Ca2+ and Mg2+ ions imparted structural stability to RfGH16_21. Dynamic light scattering analysis of RfGH16_21 showed monodispersity and hydrodynamic radius of 4.0 nm at 2.0 mg/mL protein concentration, which was comparable with the radius of gyration of 3.2 nm determined by MD simulation showing the protein to be in monomeric form. Communicated by Ramaswamy H. Sarma

Extracellular production of a thermostable Cellvibrio endolytic β-agarase in Escherichia coli for agarose liquefaction

Four GH16 family β-agarases (GH16A, GH16B, GH16C, and GH16D), originated from an agarolytic bacterium Cellvibrio sp. KY-GH-1, were expressed in an Escherichia coli system and their activities were compared. Only GH16B (597 amino acids, 63.8 kDa), with N-terminal 22-amino acid signal sequence, was secreted into the culture supernatant and demonstrated a robust endolytic agarose hydrolyzing activity for producing neoagarotetraose (NA4) and neoagarohexaose (NA6) as end products. The optimal temperature and pH for the enzyme activity were 50 °C and 7.0, respectively. The enzyme was stable up to 50 °C and over a pH range of 5.0–8.0. The kinetic parameters, including Km, Vmax, kcat, and kcat/Km, of GH16B β-agarases for agarose were 14.40 mg/mL, 542.0 U/mg, 576.3 s−1, and 4.80 × 106 s−1 M−1, respectively. The addition of 1 mM MnCl2 and 15 mM tris(2-carboxyethyl)phosphine enhanced the enzymatic activity. When agarose or neoagaro-oligosaccharides were used as substrates, the end products of enzymatic catalysis were NA4 and NA6, whereas agaropentaose was produced along with NA4 and NA6 when agaro-oligosaccharides were used as substrates. Treatment of 9%[w/v] melted agarose with the enzyme (1.6 µg/mL) under continuous magnetic stirring at 50 °C for 14 h resulted in efficient agarose liquefaction into NA4 and NA6. Purification of NA4 and NA6 from the enzymatic hydrolysate (9%[w/v] agarose, 20 mL) via Sephadex G-15 column chromatography yielded ~ 650 mg NA4/~ 900 mg NA6 (i.e., ~ 85.3% of the theoretical maximum yield). These findings suggest that the recombinant thermostable GH16B β-agarase is useful for agarose liquefaction to produce NA4 and NA6.Graphical

An insight into microbial sources, classification and industrial applications of xylanases: A rapid review.

Endo 1,4-β-d- xylanases (EC3.2.1.8) are one of the key lignocellulose hydrolyzing enzymes. Xylan, which is present in copious amounts on earth, forms the primary substrate of endo-xylanases which can unchain the constituent monosaccharides linked via β-1,4 glycosidic bonds from the xylan backbone. Researchers have shown keen interest in the xylanases belonging to glycoside hydrolase families 10 and 11 whereas those placed in other GH families are yet to be investigated. Various microbes such as bacteria and fungi harbor these enzymes for the metabolism of their lignocellulose fibers. These microbes can be used as miniature biofactories of xylanase enzymes for a plethora of environmentally benign applications in pulp and paper industry, biofuel production and for improving the quality of food in bread baking and fruit juice industry. This review highlights the potential of microbes in production of xylanase for industrial biotechnology. This article is protected by copyright. All rights reserved.

Non-specific effects of the CINNAMATE-4-HYDROXYLASE inhibitor piperonylic acid.

Chemical inhibitors are often implemented for the functional characterization of genes to overcome the limitations associated with genetic approaches. Although it is well established that the specificity of the compound is key to success of a pharmacological approach, off-target effects are often overlooked or simply neglected in a complex biological setting. Here we illustrate the cause and implications of such secondary effects by focusing on piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H) that is frequently used to investigate the involvement of lignin during plant growth and development. When supplied to plants, we found that PA is recognized as a substrate by GRETCHEN HAGEN 3.6 (GH3.6), an amido synthetase involved in the formation of the indole-3-acetic acid (IAA) conjugate IAA-Asp. By competing for the same enzyme, PA interferes with IAA conjugation, resulting in an increase in IAA concentrations in the plant. In line with the broad substrate specificity of the GH3 family of enzymes, treatment with PA increased not only IAA levels but also those of other GH3-conjugated phytohormones, namely jasmonic acid and salicylic acid. Finally, we found that interference with the endogenous function of GH3s potentially contributes to phenotypes previously observed upon PA treatment. We conclude that deregulation of phytohormone homeostasis by surrogate occupation of the conjugation machinery in the plant is likely a general phenomenon when using chemical inhibitors. Our results hereby provide a novel and important basis for future reference in studies using chemical inhibitors.

Molecular Cloning and Expression Analysis of the Typical Class III Chitinase Genes from Three Mangrove Species under Heavy Metal Stress.

Chitinases are considered to act as defense proteins when plants are exposed to heavy metal stresses. Typical class III chitinase genes were cloned from Kandelia obovate, Bruguiera gymnorrhiza, and Rhizophora stylosa by using RT-PCR and RACE and named KoCHI III, BgCHI III, and RsCHI III. Bioinformatics analysis revealed that the three genes encoding proteins were all typical class III chitinases with the characteristic catalytic structure belonging to the family GH18 and located outside the cell. In addition, there are heavy metal binding sites in the three-dimensional spatial structure of the type III chitinase gene. Phylogenetic tree analysis indicated that CHI had the closest relationship with chitinase in Rhizophora apiculata. In mangrove plants, the balance of the oxidative system in the body is disrupted under heavy metal stress, resulting in increased H2O2 content. Real-time PCR illustrated that the expression level under heavy metal stress was significantly higher than that in the control group. Expression levels of CHI III were higher in K. obovate than in B. gymnorrhiza and R. stylosa. With the increase in heavy metal stress time, the expression level increased continuously. These results suggest that chitinase plays an important role in improving the heavy metal tolerance of mangrove plants.

In Silico Analysis of a GH3 β-Glucosidase from Microcystis aeruginosa CACIAM 03.

Cyanobacteria are rich sources of secondary metabolites and have the potential to be excellent industrial enzyme producers. β-glucosidases are extensively employed in processing biomass degradation as they mediate the most crucial step of bioconversion of cellobiose (CBI), hence controlling the efficiency and global rate of biomass hydrolysis. However, the production and availability of these enzymes derived from cyanobacteria remains limited. In this study, we evaluated the β-glucosidase from Microcystis aeruginosa CACIAM 03 (MaBgl3) and its potential for bioconversion of cellulosic biomass by analyzing primary/secondary structures, predicting physicochemical properties, homology modeling, molecular docking, and simulations of molecular dynamics (MD). The results showed that MaBgl3 derives from an N-terminal domain folded as a distorted β-barrel, which contains the conserved His-Asp catalytic dyad often found in glycosylases of the GH3 family. The molecular docking results showed relevant interactions with Asp81, Ala271 and Arg444 residues that contribute to the binding process during MD simulation. Moreover, the MD simulation of the MaBgl3 was stable, shown by analyzing the root mean square deviation (RMSD) values and observing favorable binding free energy in both complexes. In addition, experimental data suggest that MaBgl3 could be a potential enzyme for cellobiose-hydrolyzing degradation.

Glycoside hydrolase family 5 gene Pp07886 in Pythium porphyrae: Identification, characterization, expression pattern, and activation of the host immunity

The oomycete Pythium porphyrae is a causative agent of red rot disease in the economically important alga Neopyropia yezoensis. However, little is known about the pathogenic mechanisms of P. porphyrae, although pathogenicity is known to involve glycoside hydrolases (GHs). GHs are involved in penetration of the host cell wall to allow oomycete invasion, as well as in nutrition and in counteracting the innate immune response of the host. In this study, a preliminary investigation of a GH5 family gene Pp07886 from P. porphyrae was performed. This gene was cloned from the P. porphyrae genome and phylogenetically identified as part of the GH5_23 subfamily. The recombinant Pp07886 (rPp07886) was found to be a polyspecific enzyme with xylanase and hesperidinase activity; thus, it might be involved in breaking down the algal cell wall and hydrolyzing hesperidin to counteract the host immune defenses. The qRT-PCR and western blot results showed that Pp07886 expression was strongly induced (187 to 1496 fold compared to vegetative mycelia) during the early infection stage (30 min to 1 day post inoculation). Furthermore, transcripts of Pp07886 were strongly induced by both xylan and hesperidin (4 and 9 fold compared to vegetative mycelia grown in SGG medium). Finally, rPp07886 was able to activate the expression of the host innate immune genes (20 to 702 fold compared to uninfected thalli cultured in PES seawater). These results suggested that Pp07886 was involved in early infection and induction of innate immunity in N. yezoensis. Thus, this research provided important data for further functional study of GH5 proteins in P. porphyrae, and increased the understanding of the molecular mechanism of pathogenicity of GH5 proteins in oomycetes.