Hydrolase Superfamily Research Articles

Search for and functional annotation of multi-domain PLA2 family proteins in flatworms

The phospholipase A2 (PLA2) is a superfamily of hydrolases that catalyze the hydrolysis of phospholipids and play a key role in many molecular processes in the cells and the organism as a whole. This family consists of 16 groups divided into six main types. PLA2 were first isolated from venom toxins and porcine pancreatic juice. The study of these enzymes is currently of great interest, since it has been shown that a number of PLA2 are involved in the processes of carcinogenesis. PLA2 enzymes were characterized in detail in model organisms and humans. However, their presence and functional role in non-model organisms is poorly understood. Such poorly studied taxa include flatworms, a number of species of which are human parasites. Several PLA2 genes have previously been characterized in parasitic flatworms and their possible role in parasite-host interaction has been shown. However, no systematic identification of the PLA2 genes in this taxon has been carried out. The paper provides a search for and a comparative analysis of PLA2 sequences encoded in the genomes of flatworms. 44 species represented by two free-living and 42 parasitic organisms were studied. The analysis was based on identification of orthologous groups of protein-coding genes, taking into account the domain structure of proteins. In flatworms, 12 of the 13 known types of animal A2 phospholipases were found, represented by 11 orthologous groups. Some phospholipases of several types fell into one orthologous group, some types split into several orthogroups in accordance with their domain structure. It has been shown that phospholipases A2 of the calcium-independent type, platelet-activating phospho­lipases from group G8 and lysosomal phospholipases from group G15 are represented in all large taxa of flatworms and the vast majority of the species studied by us. In free-living flatworms PLA2 genes have multiple copies. In parasitic flatworms, on the contrary, loss of genes occur specifically in individual taxa specifically for groups or sub­families of PLAs. An orthologous group of secreted phospholipases has been identified, which is represented only in Digenea and this family has undergone duplications in the genomes of opisthorchids. Interestingly, a number of experimental studies have previously shown the effect of Clonorchis sinensis proteins of this orthogroup on the cancer transformation of host cells. Our results made it possible for the first time to systematically identify PLA2 sequences in flatworms, and demonstrated that their evolution is subject to gene loss processes characteristic of parasite genomes in general. In addition, our analysis allowed us to identify taxon-specific processes of duplication and loss of PLA2 genes in parasitic organisms, which may be associated with the processes of their interaction with the host organism.

GWAS analysis for plant height and stem diameter in sorghum using multiple phenotyping approaches

AbstractSorghum (Sorghum bicolor (L.) Moench) is an important cereal crop cultivated around the globe. To identify the genomic regions responsible for plant height and stem diameter, a subset of the sorghum association panel was phenotyped using manual, ground‐robot, and drone‐based phenotyping approaches during 2019 and 2020 at Tifton, GA. Manual and ground‐robot‐based plant heights had a lower correlation (r = 0.55) than the manual and drone‐based measurements (r = 0.61). Tukey's mean difference plot analysis indicated that both high‐throughput methods were less accurate with taller accessions. In genome‐wide association studies using single nucleotide polymorphisms (SNPs), insertions and deletions (indels), and copy number variants, a total of 136 and 18 SNP loci associated with plant height were identified using mixed linear model and fixed and random model circulating probability unification models. For the first time, we report eight significant SNPs associated with stem diameter using a ground‐based robot. We were able to detect all four major dwarfing genes (Dw1 through Dw4) using manual phenotyping; in contrast, Dw2 was identified using both manual and ground‐robot‐based phenotyping. Sorbi.3004G093400, a gene located 19 kb upstream of the SNP locus SBI‐04_7987371 and a member of the glycoside hydrolase superfamily 35, plays a role in stem diameter, plant height, and is involved in cellular processes. Novel associations (eight for stem diameter), SNPs, and structural variants identified in this study can be used for manipulating plant height and stem diameter in sorghum.

Regulation Roles of Juvenile Hormone Epoxide Hydrolase Gene 2 in the Female River Prawn Macrobrachium nipponense Reproductive Process.

In this study, we investigated the regulatory roles of the juvenile hormone epoxide hydrolase (JHEH) gene in the reproductive process of female Macrobrachium nipponense. Its total cDNA length was 1848 bp, encoding for 460 amino acids. It contained conserved domains typical of epoxide hydrolases, such as the Abhydrolase family domain, the EHN epoxide hydrolase superfamily domain, and the "WWG" and "HGWP" motifs. The qPCR results showed that the expression of Mn-JHEH was the highest in hepatopancreas. Mn-JHEH was expressed at all stages of the embryonic and larval stages. The expression of Mn-JHEH at different developmental periods of the ovary was positively correlated with ovarian maturation. In situ hybridization showed that it was mainly located in the cytoplasmic membrane and nucleus of oocytes. The RNA interference technique was used to study the role of Mn-JHEH in the process of ovarian maturation. The knockdown of Mn-JHEH with dsRNA in the experimental group resulted in a significant decrease in the percentage of ovaries exceeding stage O-III and the gonadal index compared with the control group. On day 14 (the second molt), the molt frequency was significantly higher in the control group than in the experimental group. The results showed that Mn-JHEH played an important role in ovarian maturation and molting.

Optimization of a high throughput screening platform to identify inhibitors of asymmetric diadenosine polyphosphatases

When stressed, cells synthesize di-adenosine polyphosphates (ApnA), and cellular organisms also express proteins that degrade these compounds to release ATP. Most of these proteins are members of the nudix hydrolase superfamily, and several are involved in bacterial pathogenesis, neurodevelopment, and cancer. The goal of this project is to assist in the discovery of inhibitors of these enzymes that could be used to study ApnA function and the cellular role of these nudix enzymes. Because these enzymes cleave Ap4A and Ap5A to produce ATP, two standard ATP detection techniques were optimized and compared here for their suitability for high throughput screening. In the first assay, cleavage is monitored by coupling to a reaction catalyzed by firefly luciferase. In the second assay, cleavage is detected by coupling to hexokinase, glucose 6-phosphate dehydrogenase, and diaphorase. Although the former assay was more sensitive, the latter was more reproducible, linear, and suitable for screening and kinetic analyses. The assays were used to characterize the kinetics of reactions catalyzed by various nudix enzymes isolated from E. coli, humans, and Mycobacterium tuberculosis, the bacterium that causes tuberculosis. Results reveal subtle differences between the proteins that might be exploited to identify specific small molecule inhibitors.

Cloning, Expression, Purification and Enzymatic Characterization of Low-temperature Cholesterol Esterase from Marine Panthenia Agglutinosa

BACKGROUND: The low-temperature cholesterol esterase is primarily used in industries such as papermaking and healthcare. OBJECTIVE: To discover a microorganism with high cholesterol esterase activity and tolerance to low temperatures, leading to the promotion of the sustainable utilization of marine cold-adapted microbial resources and fostering industrial development. MATERIALS AND METHODS: This study isolated a strain producing low-temperature cholesterol esterase from marine samples in the China Bohai Sea. The strain was identified through 16S rDNA sequencing and named Panthenia agglutinosa Y03. The cholesterol esterase gene (PaChe) from P. agglutinosa Y03 was cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme PaChe was purified and characterized. The structure of PaChe was predicted using AlphaFold2, and molecular docking was performed with cholesterol linoleate as the ligand. RESULTS: The enzyme protein has a molecular weight of 56.35 KDa, a theoretical pI of 7.24, lacks a signal peptide, and exhibits structural features of the α/β hydrolase superfamily protein. The concentration of the purified PaChe is 0.5 mg/mL, with a specific activity of 42.7 U/mg. The optimal working temperature is 30 °C, and the enzyme retains activity at 4 °C , demonstrating weaker thermal stability. The optimal pH is 7, and the enzyme maintains over 70% activity at pH 9. Na +, Ca 2+ and Mg 2+ are the primary activators, while Ba 2+, Fe 2+, Mn 2+, Cu 2+ and chemical agents such as SDS as inhibitors, with Cu2+ exhibiting particularly significant inhibitory effects. CONCLUSION: This study establishes the theoretical groundwork for the development and utilization of a novel lowtemperature cholesterol esterase.

Development of Tunable Mechanism-Based Carbasugar Ligands that Stabilize Glycoside Hydrolases through the Formation of Transient Covalent Intermediates.

Mutations in many members of the set of human lysosomal glycoside hydrolases cause a wide range of lysosomal storage diseases. As a result, much effort has been directed toward identifying pharmacological chaperones of these lysosomal enzymes. The majority of the candidate chaperones are active site-directed competitive iminosugar inhibitors but these have met with limited success. As a first step toward an alternative class of pharmacological chaperones we explored the potential of small molecule mechanism-based reversible covalent inhibitors to form transient enzyme-inhibitor adducts. By serial synthesis and kinetic analysis of candidate molecules, we show that rational tuning of the chemical reactivity of glucose-configured carbasugars delivers cyclohexenyl-based allylic carbasugar that react with the lysosomal enzyme β-glucocerebrosidase (GCase) to form covalent enzyme-adducts with different half-lives. X-ray structural analysis of these compounds bound noncovalently to GCase, along with the structures of the covalent adducts of compounds that reacted with the catalytic nucleophile of GCase, reveal unexpected reactivities of these compounds. Using differential scanning fluorimetry, we show that formation of a transient covalent intermediate stabilizes the folded enzyme against thermal denaturation. In addition, these covalent adducts break down to liberate the active enzyme and a product that is no longer inhibitory. We further show that the one compound, which reacts through an unprecedented SN1'-like mechanism, exhibits exceptional reactivity-illustrated by this compound also covalently labeling an α-glucosidase. We anticipate that such carbasugar-based single turnover covalent ligands may serve as pharmacological chaperones for lysosomal glycoside hydrolases and other disease-associated retaining glycosidases. The unusual reactivity of these molecules should also open the door to creation of new chemical biology probes to explore the biology of this important superfamily of glycoside hydrolases.

Structural Analysis of Mammalian Sialic Acid Esterase

Sialic acid esterase (SIAE) catalyzes the removal of O-acetyl groups from sialic acids found on cell surface glycoproteins to regulate cellular processes such as B cell receptor signalling and apoptosis. Loss-of-function mutations in SIAE are associated with several common autoimmune diseases including Crohn’s, ulcerative colitis, and arthritis. To gain a better understanding of the function and regulation of this protein, we determined crystal structures of SIAE from three mammalian homologs, including an acetate bound structure. The structures reveal that the catalytic domain adopts the fold of the SGNH hydrolase superfamily. The active site is composed of a catalytic dyad, as opposed to the previously reported catalytic triad. Attempts to determine a substrate-bound structure yielded only the hydrolyzed product acetate in the active site. Rigid docking of complete substrates followed by molecular dynamics simulations revealed that the active site does not form specific interactions with substrates, rather it appears to be broadly specific to accept sialoglycans with diverse modifications. Based on the acetate bound structure, a catalytic mechanism is proposed. Structural mapping of disease mutations reveals that most are located on the surface of the enzyme and would only cause minor disruptions to the protein fold, suggesting that these mutations likely affect binding to other factors. These results improve our understanding of SIAE biology and may aid in the development of therapies for autoimmune diseases and cancer.

Biodegradation of polybutylene succinate by an extracellular esterase from Pseudomonas mendocina

An extracellular esterase (HP) with polybutylene succinate (PBS)-degrading ability was identified from Pseudomonas mendocina SA-1503. The HP also had the ability to degrade poly(3-hydroxybutyrate-co-4-hydroxybutyrate) and polycaprolactone. This HP had optimal activity at pH 9.0 and 40 °C and remained stable at pH 8.0–9.0 and temperatures of 30–40 °C. Mn2+ promoted the enzyme activity. HP could hydrolyze all p-NP fatty acid ester substrates containing even numbers of carbon atoms from C2 to C18 and had the highest catalytic activity for the p-NP C6 substrate. After 60 h of HP-catalyzed degradation, PBS films experienced a weight loss of more than 60%. Butanedioic acid, 1,4-butanediol, and a series of oligomers were detected in the degradation products of PBS by HP. Further structural analysis of HP revealed that it could be classified as a microbial esterase of α/β hydrolase superfamily and contained a conserved catalytic triad structure (Ser-148, Asp-198, and His-228) with a relatively exposed active site.

Gene cloning, IPTG-independent auto-induction and characterization of a novel hyperstable S9 prolyl oligopeptidase having lipolytic activity from Thermotoga naphthophila RKU-10T with applications

A hyperstable lipase from Thermotoga naphthophila (TnLip) was cloned and overexpressed as a soluble and active monomeric protein in an effectual mesophilic host system. Sequence study revealed that TnLip is a peptidase S9 prolyl oligopeptidase domain (acetyl esterase/lipase-like protein), belongs to alpha/beta (α/β)-hydrolase superfamily containing a well-conserved α/β-hydrolase fold and penta-peptide (GLSAG) motif. Various cultivation and induction strategies were applied to improve the heterologous expression and bacterial biomass, but TnLip intracellular activity was enhanced by 14.25- fold with IPTG-independent auto-induction approach after 16 h (26 °C, 150 rev min−1) incubation. Purified TnLip (35 kDa) showed peak activity at 85 °C in McIlvaine buffer (pH 7.0–8.0), and has great stability over a broad range of pH (5.0–10.0), and temperature (40–85 °C) for 8 h. TnLip exhibited prodigious resistance toward various commercial detergents, chemical additives, and salt. TnLip activity was improved by 170.51 %, 130.67 %, 127.42 %, 126.54 %, 126.61 %, 120.32 %, and 116.31 % with 50 % (v/v) of methanol, ethanol, n-butanol, isopropanol, acetone, glycerol, and acetic acid, respectively. Moreover, with 3.0 M of NaCl, and 10 mM of Ca2+, Mn2+, and Mg2+ TnLip activity was augmented by 210 %, 185.64 %, 152.03 %, and 116.26 %, respectively. TnLip has an affinity with various substrates (p-nitrophenyl ester and natural oils) but maximal hydrolytic activity was perceived with p-nitrophenyl palmitate (pNPP, 3600 U mg−1) and olive oil (1182.05 U mg−1). The values of Km (0.576 mM), Vmax (4216 μmol mg−1 min−1), VmaxKm−1 (7319.44 min−1), kcat (1106.74 s−1), and kcatKm−1 (1921.42 mM−1 s−1) were calculated using pNPP substrate. Additionally, TnLip degraded animals' fats and removed oil stains within 3 h and 5 min, respectively. All these features make halo-alkali-thermophilic TnLip as an auspicious contender for laundry detergents (cleaning bio-additive), fat degradation, wastewater treatment and endorse eco-friendly stewardship along with various other biotechnological applications.

The global distribution of the macrolide esterase EstX from the alpha/beta hydrolase superfamily

Macrolide antibiotics, pivotal in clinical therapeutics, are confronting resistance challenges mediated by enzymes like macrolide esterases, which are classified into Ere-type and the less studied Est-type. In this study, we provide the biochemical confirmation of EstX, an Est-type macrolide esterase that initially identified as unknown protein in the 1980s. EstX is capable of hydrolyzing four 16-membered ring macrolides, encompassing both veterinary (tylosin, tidipirosin, and tilmicosin) and human-use (leucomycin A5) antibiotics. It uses typical catalytic triad (Asp233-His261-Ser102) from alpha/beta hydrolase superfamily for ester bond hydrolysis. Further genomic context analysis suggests that the dissemination of estX is likely facilitated by mobile genetic elements such as integrons and transposons. The global distribution study indicates that bacteria harboring the estX gene, predominantly pathogenic species like Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae, are prevalent in 74 countries across 6 continents. Additionally, the emergence timeline of the estX gene suggests its proliferation may be linked to the overuse of macrolide antibiotics. The widespread prevalence and dissemination of Est-type macrolide esterase highlight an urgent need for enhanced monitoring and in-depth research, underlining its significance as an escalating public health issue.

Genome-wide identification and mining elite allele variation of the Monoacylglycerol lipase (MAGL) gene family in upland cotton (Gossypium hirsutum L.)

BackgroundMonoacylglycerol lipase (MAGL) genes belong to the alpha/beta hydrolase superfamily, catalyze the terminal step of triglyceride (TAG) hydrolysis, converting monoacylglycerol (MAG) into free fatty acids and glycerol.ResultsIn this study, 30 MAGL genes in upland cotton have been identified, which have been classified into eight subgroups. The duplication of GhMAGL genes in upland cotton was predominantly influenced by segmental duplication events, as revealed through synteny analysis. Furthermore, all GhMAGL genes were found to contain light-responsive elements. Through comprehensive association and haplotype analyses using resequencing data from 355 cotton accessions, GhMAGL3 and GhMAGL6 were detected as key genes related to lipid hydrolysis processes, suggesting a negative regulatory effect.ConclusionsIn summary, MAGL has never been studied in upland cotton previously. This study provides the genetic mechanism foundation for the discover of new genes involved in lipid metabolism to improve cottonseed oil content, which will provide a strategic avenue for marker-assisted breeding aimed at incorporating desirable traits into cultivated cotton varieties.

Pyrazolone compounds could inhibit CES1 and ameliorates fat accumulation during adipocyte differentiation

Carboxylesterase 1 (CES1), a member of the serine hydrolase superfamily, is involved in a wide range of xenobiotic and endogenous substances metabolic reactions in mammals. The inhibition of CES1 could not only alter the metabolism and disposition of related drugs, but also be benefit for treatment of metabolic disorders, such as obesity and fatty liver disease. In the present study, we aim to develop potential inhibitors of CES1 and reveal the preferred inhibitor structure from a series of synthetic pyrazolones (compounds 1–27). By in vitro high-throughput screening method, we found compounds 25 and 27 had non-competitive inhibition on CES1-mediated N-alkylated d-luciferin methyl ester (NLMe) hydrolysis, while compound 26 competitively inhibited CES1-mediated NLMe hydrolysis. Additionally, Compounds 25, 26 and 27 can inhibit CES1-mediated fluorescent probe hydrolysis in live HepG2 cells with effect. Besides, compounds 25, 26 and 27 could effectively inhibit the accumulation of lipid droplets in mouse adipocytes cells. These data not only provided study basis for the design of newly CES1 inhibitors. The present study not only provided the basis for the development of lead compounds for novel CES1 inhibitors with better performance, but also offered a new direction for the explore of candidate compounds for the treatment of hyperlipidemia and related diseases.

Discovery and mechanism explanation of a novel green biocatalyst esterase Bur01 from Burkholderia ambifaria for ester synthesis under aqueous phase

Biocatalyst catalyzing the synthesis of esters under aqueous phase is an alternative with green and sustainable characteristics. Here, a biocatalyst esterase Bur01 was identified through genome sequencing and gene library construction from a Burkholderia ambifaria BJQ0010 with efficient ester synthesis property under aqueous phase for the first time. Bur01 was soluble expressed and the purified enzyme showed the highest activity at pH 4.0 and 40 °C. It had a broad substrate spectrum, especially for ethyl esters. The structure of Bur01 was categorized as a member of α/β fold hydrolase superfamily. The easier opening of lid under aqueous phase and the hydrophobicity of substrate channel contribute to easier access to the active center for substrate. Molecular docking and site-directed mutation demonstrated that the oxyanion hole Ala22, Met112 and π-bond stacking between His24 and Phe217 played essential roles in catalytic function. The mutants V149A, V149I, L159I and F137I enhanced enzyme activity to 1.42, 1.14, 1.32 and 2.19 folds due to reduced spatial resistance and increased hydrophobicity of channel and ethyl octanoate with the highest conversion ratio of 68.28 % was obtained for F137I. These results provided new ideas for developing green catalysts and catalytic basis of mechanistic studies for ester synthetase under aqueous phase.

ATP Hydrolases Superfamily Protein 1 (ASP1) Maintains Root Stem Cell Niche Identity through Regulating Reactive Oxygen Species Signaling in Arabidopsis.

The maintenance of the root stem cell niche identity in Arabidopsis relies on the delicate balance of reactive oxygen species (ROS) levels in root tips; however, the intricate molecular mechanisms governing ROS homeostasis within the root stem cell niche remain unclear. In this study, we unveil the role of ATP hydrolase superfamily protein 1 (ASP1) in orchestrating root stem cell niche maintenance through its interaction with the redox regulator cystathionine β-synthase domain-containing protein 3 (CBSX3). ASP1 is exclusively expressed in the quiescent center (QC) cells and governs the integrity of the root stem cell niche. Loss of ASP1 function leads to enhanced QC cell division and distal stem cell differentiation, attributable to reduced ROS levels and diminished expression of SCARECROW and SHORT ROOT in root tips. Our findings illuminate the pivotal role of ASP1 in regulating ROS signaling to maintain root stem cell niche homeostasis, achieved through direct interaction with CBSX3.

An exchange of single amino acid between the phosphohydrolase modules of Escherichia coli MutT and Mycobacterium smegmatis MutT1 switches their cleavage specificities

MutT proteins belong to the Nudix hydrolase superfamily that includes a diverse group of Mg2+ requiring enzymes. These proteins use a generalized substrate, nucleoside diphosphate linked to a chemical group X (NDP-X), to produce nucleoside monophosphate (NMP) and the moiety X linked with phosphate (XP). E. coli MutT (EcoMutT) and mycobacterial MutT1 (MsmMutT1) belong to the Nudix hydrolase superfamily that utilize 8-oxo-(d)GTP (referring to both 8-oxo-GTP or 8-oxo-dGTP). However, predominant products of their activities are different. While EcoMutT produces 8-oxo-(d)GMP, MsmMutT1 gives rise to 8-oxo-(d)GDP. Here, we show that the altered cleavage specificities of the two proteins are largely a consequence of the variation at the equivalent of Gly37 (G37) in EcoMutT to Lys (K65) in the MsmMutT1. Remarkably, mutations of G37K (EcoMutT) and K65G (MsmMutT1) switch their cleavage specificities to produce 8-oxo-(d)GDP, and 8-oxo-(d)GMP, respectively. Further, a time course analysis using 8-oxo-GTP suggests that MsmMutT1(K65G) hydrolyses 8-oxo-(d)GTP to 8-oxo-(d)GMP in a two-step reaction via 8-oxo-(d)GDP intermediate. Expectedly, unlike EcoMutT (G37K) and MsmMutT1, EcoMutT and MsmMutT1 (K65G) rescue an E. coli ΔmutT strain, better by decreasing A to C mutations.

Abstract 1921 Crystallization of a Diadenosine Polyphosphatase of the Nudix Hydrolase Superfamily from M. tuberculosis

Abstract 1921 Crystallization of a Diadenosine Polyphosphatase of the Nudix Hydrolase Superfamily from M. tuberculosis

Metabolism of host lysophosphatidylcholine in Plasmodium falciparum–infected erythrocytes

The human malaria parasite Plasmodium falciparum requires exogenous fatty acids to support its growth during the pathogenic, asexual erythrocytic stage. Host serum lysophosphatidylcholine (LPC) is a significant fatty acid source, yet the metabolic processes responsible for the liberation of free fatty acids from exogenous LPC are unknown. Using an assay for LPC hydrolysis in P. falciparum-infected erythrocytes, we have identified small-molecule inhibitors of key in situ lysophospholipase activities. Competitive activity-based profiling and generation of a panel of single-to-quadruple knockout parasite lines revealed that two enzymes of the serine hydrolase superfamily, termed exported lipase (XL) 2 and exported lipase homolog (XLH) 4, constitute the dominant lysophospholipase activities in parasite-infected erythrocytes. The parasite ensures efficient exogenous LPC hydrolysis by directing these two enzymes to distinct locations: XL2 is exported to the erythrocyte, while XLH4 is retained within the parasite. While XL2 and XLH4 were individually dispensable with little effect on LPC hydrolysis in situ, loss of both enzymes resulted in a strong reduction in fatty acid scavenging from LPC, hyperproduction of phosphatidylcholine, and an enhanced sensitivity to LPC toxicity. Notably, growth of XL/XLH-deficient parasites was severely impaired when cultured in media containing LPC as the sole exogenous fatty acid source. Furthermore, when XL2 and XLH4 activities were ablated by genetic or pharmacologic means, parasites were unable to proliferate in human serum, a physiologically relevant fatty acid source, revealing the essentiality of LPC hydrolysis in the host environment and its potential as a target for anti-malarial therapy.

Marker and readout genes for defense priming in Pseudomonas cannabina pv. alisalensis interaction aid understanding systemic immunity in Arabidopsis

Following localized infection, the entire plant foliage becomes primed for enhanced defense. However, specific genes induced during defense priming (priming-marker genes) and those showing increased expression in defense-primed plants upon rechallenge (priming-readout genes) remain largely unknown. In our Arabidopsis thaliana study, genes AT1G76960 (function unknown), CAX3 (encoding a vacuolar Ca2+/H+ antiporter), and CRK4 (encoding a cysteine-rich receptor-like protein kinase) were strongly expressed during Pseudomonas cannabina pv. alisalensis-induced defense priming, uniquely marking the primed state for enhanced defense. Conversely, PR1 (encoding a pathogenesis-related protein), RLP23 and RLP41 (both encoding receptor-like proteins) were similarly activated in defense-primed plants before and after rechallenge, suggesting they are additional marker genes for defense priming. In contrast, CASPL4D1 (encoding Casparian strip domain-like protein 4D1), FRK1 (encoding flg22-induced receptor-like kinase), and AT3G28510 (encoding a P loop-containing nucleoside triphosphate hydrolases superfamily protein) showed minimal activation in uninfected, defense-primed, or rechallenged plants, but intensified in defense-primed plants after rechallenge. Notably, mutation in only priming-readout gene NHL25 (encoding NDR1/HIN1-like protein 25) impaired both defense priming and systemic acquired resistance, highlighting its previously undiscovered pivotal role in systemic plant immunity.

Functional characterization and structural insights of three cutinases from the ascomycete Fusarium verticillioides

Cutinases are serine esterases that belong to the α/β hydrolases superfamily. The natural substrates for these enzymes are cutin and suberin, components of the plant cuticle, the first barrier in the defense system against pathogen invasion. It is well-reported that plant pathogens produce cutinases to facilitate infection. Fusarium verticillioides, one important corn pathogens, is an ascomycete upon which its cutinases are poorly explored. Consequently, the objective of this study was to perform the biochemical characterization of three precursor cutinases (FvCut1, FvCut2, and FvCut3) from F. verticillioides and to obtain structural insights about them. The cutinases were produced in Escherichia coli and purified. FvCut1, FvCut2, and FvCut3 presented optimal temperatures of 20, 40, and 35 °C, and optimal pH of 9, 7, and 8, respectively. Some chemicals stimulated the enzymatic activity. The kinetic parameters revealed that FvCut1 has higher catalytic efficiency (Kcat/Km) in the p-nitrophenyl-butyrate (p-NPB) substrate. Nevertheless, the enzymes were not able to hydrolyze polyethylene terephthalate (PET). Furthermore, the three-dimensional models of these enzymes showed structural differences among them, mainly FvCut1, which presented a narrower opening cleft to access the catalytic site. Therefore, our study contributes to exploring the diversity of fungal cutinases and their potential biotechnological applications.

Infection of postharvest pear by Penicillium expansum is facilitated by the glycoside hydrolase (eglB) gene

The primary reason for postharvest loss is blue mold disease which is mainly caused by Penicillium expansum. Strategies for disease control greatly depend on the understanding of mechanisms of pathogen-fruit interaction. A member of the glycoside hydrolase family, β-glucosidase 1b (eglB), in P. expansum was significantly upregulated during postharvest pear infection. Glycoside hydrolases are a large group of enzymes that can degrade plant cell wall polymers. High homology was found between the glycoside hydrolase superfamily in P. expansum. Functional characterization and analysis of eglB were performed via gene knockout and complementation analysis. Although eglB deletion had no notable effect on P. expansum colony shape or microscopic morphology, it did reduce the production of fungal hyphae, thereby reducing P. expansum's sporulation and patulin (PAT) accumulation. Moreover, the deletion of eglB (ΔeglB) reduced P. expansum pathogenicity in pears. The growth, conidia production, PAT accumulation, and pathogenicity abilities of ΔeglB were restored to that of wild-type P. expansum by complementation of eglB (ΔeglB-C). These findings indicate that eglB contributes to P. expansum's development and pathogenicity. This research is a contribution to the identification of key effectors of fungal pathogenicity for use as targets in fruit safety strategies.