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1.
Commun Biol ; 7(1): 574, 2024 May 15.
Article in English | MEDLINE | ID: mdl-38750105

ABSTRACT

Metastases are the major cause of cancer-related death, yet, molecular weaknesses that could be exploited to prevent tumor cells spreading are poorly known. Here, we found that perturbing hydrolase transport to lysosomes by blocking either the expression of IGF2R, the main receptor responsible for their trafficking, or GNPT, a transferase involved in the addition of the specific tag recognized by IGF2R, reduces melanoma invasiveness potential. Mechanistically, we demonstrate that the perturbation of this traffic, leads to a compensatory lysosome neo-biogenesis devoided of degradative enzymes. This regulatory loop relies on the stimulation of TFEB transcription factor expression. Interestingly, the inhibition of this transcription factor playing a key role of lysosome production, restores melanomas' invasive potential in the absence of hydrolase transport. These data implicate that targeting hydrolase transport in melanoma could serve to develop new therapies aiming to prevent metastasis by triggering a physiological response stimulating TFEB expression in melanoma.


Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Hydrolases , Lysosomes , Melanoma , Humans , Melanoma/genetics , Melanoma/pathology , Melanoma/metabolism , Lysosomes/metabolism , Hydrolases/metabolism , Hydrolases/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Cell Line, Tumor , Receptor, IGF Type 2/metabolism , Receptor, IGF Type 2/genetics , Neoplasm Metastasis , Protein Transport , Gene Expression Regulation, Neoplastic
2.
Int J Mol Sci ; 25(9)2024 May 04.
Article in English | MEDLINE | ID: mdl-38732240

ABSTRACT

Methicillin-resistant Staphylococcus aureus (MRSA) infection has rapidly spread through various routes. A genomic analysis of clinical MRSA samples revealed an unknown protein, Sav2152, predicted to be a haloacid dehalogenase (HAD)-like hydrolase, making it a potential candidate for a novel drug target. In this study, we determined the crystal structure of Sav2152, which consists of a C2-type cap domain and a core domain. The core domain contains four motifs involved in phosphatase activity that depend on the presence of Mg2+ ions. Specifically, residues D10, D12, and D233, which closely correspond to key residues in structurally homolog proteins, are responsible for binding to the metal ion and are known to play critical roles in phosphatase activity. Our findings indicate that the Mg2+ ion known to stabilize local regions surrounding it, however, paradoxically, destabilizes the local region. Through mutant screening, we identified D10 and D12 as crucial residues for metal binding and maintaining structural stability via various uncharacterized intra-protein interactions, respectively. Substituting D10 with Ala effectively prevents the interaction with Mg2+ ions. The mutation of D12 disrupts important structural associations mediated by D12, leading to a decrease in the stability of Sav2152 and an enhancement in binding affinity to Mg2+ ions. Additionally, our study revealed that D237 can replace D12 and retain phosphatase activity. In summary, our work uncovers the novel role of metal ions in HAD-like phosphatase activity.


Subject(s)
Bacterial Proteins , Hydrolases , Magnesium , Phosphoric Monoester Hydrolases , Magnesium/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Phosphoric Monoester Hydrolases/metabolism , Phosphoric Monoester Hydrolases/chemistry , Phosphoric Monoester Hydrolases/genetics , Hydrolases/metabolism , Hydrolases/chemistry , Hydrolases/genetics , Models, Molecular , Methicillin-Resistant Staphylococcus aureus/enzymology , Methicillin-Resistant Staphylococcus aureus/genetics , Staphylococcus aureus/enzymology , Crystallography, X-Ray , Protein Binding
3.
Proc Natl Acad Sci U S A ; 121(21): e2322501121, 2024 May 21.
Article in English | MEDLINE | ID: mdl-38748578

ABSTRACT

Biological regulation often depends on reversible reactions such as phosphorylation, acylation, methylation, and glycosylation, but rarely halogenation. A notable exception is the iodination and deiodination of thyroid hormones. Here, we report detection of bromotyrosine and its subsequent debromination during Drosophila spermatogenesis. Bromotyrosine is not evident when Drosophila express a native flavin-dependent dehalogenase that is homologous to the enzyme responsible for iodide salvage from iodotyrosine in mammals. Deletion or suppression of the dehalogenase-encoding condet (cdt) gene in Drosophila allows bromotyrosine to accumulate with no detectable chloro- or iodotyrosine. The presence of bromotyrosine in the cdt mutant males disrupts sperm individualization and results in decreased fertility. Transgenic expression of the cdt gene in late-staged germ cells rescues this defect and enhances tolerance of male flies to bromotyrosine. These results are consistent with reversible halogenation affecting Drosophila spermatogenesis in a process that had previously eluded metabolomic, proteomic, and genomic analyses.


Subject(s)
Drosophila Proteins , Fertility , Spermatogenesis , Tyrosine , Animals , Male , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Tyrosine/metabolism , Tyrosine/analogs & derivatives , Drosophila melanogaster/metabolism , Drosophila melanogaster/genetics , Drosophila/genetics , Drosophila/metabolism , Animals, Genetically Modified , Hydrolases/metabolism , Hydrolases/genetics
4.
J Hazard Mater ; 472: 134532, 2024 Jul 05.
Article in English | MEDLINE | ID: mdl-38749251

ABSTRACT

Polyethylene terephthalate (PET) is widely used for various industrial applications. However, owing to its extremely slow breakdown rate, PET accumulates as plastic trash, which negatively affects the environment and human health. Here, we report two novel PET hydrolases: PpPETase from Pseudomonas paralcaligenes MRCP1333, identified in human feces, and ScPETase from Streptomyces calvus DSM 41452. These two enzymes can decompose various PET materials, including semicrystalline PET powders (Cry-PET) and low-crystallinity PET films (gf-PET). By structure-guided engineering, two variants, PpPETaseY239R/F244G/Y250G and ScPETaseA212C/T249C/N195H/N243K were obtained that decompose Cry-PET 3.1- and 1.9-fold faster than their wild-type enzymes, respectively. The co-expression of ScPETase and mono-(2-hydroxyethyl) terephthalate hydrolase from Ideonella sakaiensis (IsMHETase) resulted in 1.4-fold more degradation than the single enzyme system. This engineered strain degraded Cry-PET and gf-PET by more than 40% and 6%, respectively, after 30 d. The concentrations of terephthalic acid (TPA) in the Cry-PET and gf-PET degradation products were 37.7% and 25.6%, respectively. The discovery of these two novel PET hydrolases provides opportunities to create more powerful biocatalysts for PET biodegradation.


Subject(s)
Feces , Hydrolases , Polyethylene Terephthalates , Streptomyces , Polyethylene Terephthalates/metabolism , Polyethylene Terephthalates/chemistry , Streptomyces/enzymology , Streptomyces/genetics , Hydrolases/metabolism , Hydrolases/genetics , Hydrolases/chemistry , Humans , Feces/microbiology , Pseudomonas/enzymology , Pseudomonas/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Burkholderiales
5.
Int J Biol Macromol ; 269(Pt 1): 132083, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38705327

ABSTRACT

Arginine deiminase (ADI) has garnered significant interest because of its ability to objectively eradicate cancer cells and produce L-citrulline. To meet the production demands, this study focused on enhancing the enzyme activity and thermal stability of ADI. In this study, 24 ADI mutants were obtained through computer aid site-specific mutation in the ADI of Enterobacter faecalis. Notably, the specific enzyme activities of F44W, N163P, E220I, E220L, N318E, A336G, T340I, and N382F increased, reaching 1.33-2.53 times that of the original enzyme. This study confirmed that site-specific mutations are critical for optimizing enzyme function. Additionally, the F44W, N163P, E220I, T340I, and A336G mutants demonstrated good thermal stability. The optimal pH for mutant F44W increased to 8, whereas mutants E220I, I244V, A336G, T340I, and N328F maintained an optimal pH of 7.5. Conversely, the M109L, N163P, E220L, I244L, and N318E mutants shad an optimal pH of 7. This study revealed that mutant enzymes with increased activity were more likely to contain mutation sites situated near the four loops associated with catalytic residues, whereas mutations at the dimer junction sites had a higher tendency to enhance enzyme stability. These findings contribute to the development of ADI industrial applications and its modifications.


Subject(s)
Enzyme Stability , Hydrolases , Hydrolases/chemistry , Hydrolases/genetics , Hydrolases/metabolism , Hydrogen-Ion Concentration , Mutation , Kinetics , Protein Engineering/methods , Biocatalysis , Mutagenesis, Site-Directed , Models, Molecular , Temperature
6.
Water Res ; 256: 121593, 2024 Jun 01.
Article in English | MEDLINE | ID: mdl-38631239

ABSTRACT

Organic contaminants enter aquatic ecosystems from various sources, including wastewater treatment plant effluent. Freshwater biofilms play a major role in the removal of organic contaminants from receiving water bodies, but knowledge of the molecular mechanisms driving contaminant biotransformations in complex stream biofilm (periphyton) communities remains limited. Previously, we demonstrated that biofilms in experimental flume systems grown at higher ratios of treated wastewater (WW) to stream water displayed an increased biotransformation potential for a number of organic contaminants. We identified a positive correlation between WW percentage and biofilm biotransformation rates for the widely-used insect repellent, N,N-diethyl-meta-toluamide (DEET) and a number of other wastewater-borne contaminants with hydrolyzable moieties. Here, we conducted deep shotgun sequencing of flume biofilms and identified a positive correlation between WW percentage and metagenomic read abundances of DEET hydrolase (DH) homologs. To test the causality of this association, we constructed a targeted metagenomic library of DH homologs from flume biofilms. We screened our complete metagenomic library for activity with four different substrates, including DEET, and a subset thereof with 183 WW-related organic compounds. The majority of active hydrolases in the metagenomic library preferred aliphatic and aromatic ester substrates while, remarkably, only a single reference enzyme was capable of DEET hydrolysis. Of the 626 total enzyme-substrate combinations tested, approximately 5% were active enzyme-substrate pairs. Metagenomic DH family homologs revealed a broad substrate promiscuity spanning 22 different compounds when summed across all enzymes tested. We biochemically characterized the most promiscuous and active enzymes identified based on metagenomic analysis from uncultivated Rhodospirillaceae and Planctomycetaceae. In addition to characterizing new DH family enzymes, we exemplified a framework for linking metagenome-guided hypothesis generation with experimental validation. Overall, this study expands the scope of known enzymatic contaminant biotransformations for metagenomic hydrolases from WW-receiving stream biofilm communities.


Subject(s)
Biofilms , Hydrolases , Wastewater , Xenobiotics , Wastewater/chemistry , Xenobiotics/metabolism , Hydrolases/metabolism , Hydrolases/genetics , Water Pollutants, Chemical/metabolism , Rivers , Biotransformation
7.
Int J Mol Sci ; 25(8)2024 Apr 09.
Article in English | MEDLINE | ID: mdl-38673749

ABSTRACT

The anticancer potential of Levilactobacillus brevis KU15176 against the stomach cancer cell line AGS has been reported previously. In this study, we aimed to analyze the genome of L. brevis KU15176 and identify key genes that may have potential anticancer properties. Among potential anticancer molecules, the role of arginine deiminase (ADI) in conferring an antiproliferative functionality was confirmed. In vitro assay against AGS cell line confirmed that recombinant ADI from L. brevis KU15176 (ADI_br, 5 µg/mL), overexpressed in E. coli BL21 (DE3), exerted an inhibitory effect on AGS cell growth, resulting in a 65.32% reduction in cell viability. Moreover, the expression of apoptosis-related genes, such as bax, bad, caspase-7, and caspase-3, as well as the activity of caspase-9 in ADI_br-treated AGS cells, was higher than those in untreated (culture medium-only) cells. The cell-scattering behavior of ADI_br-treated cells showed characteristics of apoptosis. Flow cytometry analyses of AGS cells treated with ADI_br for 24 and 28 h revealed apoptotic rates of 11.87 and 24.09, respectively, indicating the progression of apoptosis in AGS cells after ADI_br treatment. This study highlights the potential of ADI_br as an effective enzyme for anticancer applications.


Subject(s)
Apoptosis , Cell Proliferation , Hydrolases , Levilactobacillus brevis , Stomach Neoplasms , Humans , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Hydrolases/metabolism , Hydrolases/genetics , Hydrolases/pharmacology , Levilactobacillus brevis/genetics , Levilactobacillus brevis/enzymology , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Recombinant Proteins/genetics , Stomach Neoplasms/pathology , Stomach Neoplasms/drug therapy , Stomach Neoplasms/genetics
8.
Sci Rep ; 14(1): 9231, 2024 04 22.
Article in English | MEDLINE | ID: mdl-38649439

ABSTRACT

This study investigated the impact of overexpressing the mitochondrial enzyme Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) in human osteosarcoma epithelial cells (U2OS) in vitro. While the downregulation or knockdown of FAHD1 has been extensively researched in various cell types, this study aimed to pioneer the exploration of how increased catalytic activity of human FAHD1 isoform 1 (hFAHD1.1) affects human cell metabolism. Our hypothesis posited that elevation in FAHD1 activity would lead to depletion of mitochondrial oxaloacetate levels. This depletion could potentially result in a decrease in the flux of the tricarboxylic acid (TCA) cycle, thereby accompanied by reduced ROS production. In addition to hFAHD1.1 overexpression, stable U2OS cell lines were established overexpressing a catalytically enhanced variant (T192S) and a loss-of-function variant (K123A) of hFAHD1. It is noteworthy that homologs of the T192S variant are present in animals exhibiting increased resistance to oxidative stress and cancer. Our findings demonstrate that heightened activity of the mitochondrial enzyme FAHD1 decreases cellular ROS levels in U2OS cells. However, these results also prompt a series of intriguing questions regarding the potential role of FAHD1 in mitochondrial metabolism and cellular development.


Subject(s)
Bone Neoplasms , Hydrolases , Mitochondria , Osteosarcoma , Reactive Oxygen Species , Humans , Bone Neoplasms/metabolism , Bone Neoplasms/genetics , Bone Neoplasms/pathology , Cell Line, Tumor , Citric Acid Cycle , Mitochondria/metabolism , Osteosarcoma/metabolism , Osteosarcoma/genetics , Osteosarcoma/pathology , Oxidative Stress , Reactive Oxygen Species/metabolism , Hydrolases/genetics , Hydrolases/metabolism
9.
Hepatol Commun ; 8(5)2024 May 01.
Article in English | MEDLINE | ID: mdl-38668730

ABSTRACT

BACKGROUND: We previously demonstrated the successful use of in vivo CRISPR gene editing to delete 4-hydroxyphenylpyruvate dioxygenase (HPD) to rescue mice deficient in fumarylacetoacetate hydrolase (FAH), a disorder known as hereditary tyrosinemia type 1 (HT1). The aim of this study was to develop an ex vivo gene-editing protocol and apply it as a cell therapy for HT1. METHODS: We isolated hepatocytes from wild-type (C57BL/6J) and Fah-/- mice and then used an optimized electroporation protocol to deliver Hpd-targeting CRISPR-Cas9 ribonucleoproteins into hepatocytes. Next, hepatocytes were transiently incubated in cytokine recovery media formulated to block apoptosis, followed by splenic injection into recipient Fah-/- mice. RESULTS: We observed robust engraftment and expansion of transplanted gene-edited hepatocytes from wild-type donors in the livers of recipient mice when transient incubation with our cytokine recovery media was used after electroporation and negligible engraftment without the media (mean: 46.8% and 0.83%, respectively; p=0.0025). Thus, the cytokine recovery medium was critical to our electroporation protocol. When hepatocytes from Fah-/- mice were used as donors for transplantation, we observed 35% and 28% engraftment for Hpd-Cas9 ribonucleoproteins and Cas9 mRNA, respectively. Tyrosine, phenylalanine, and biochemical markers of liver injury normalized in both Hpd-targeting Cas9 ribonucleoprotein and mRNA groups independent of induced inhibition of Hpd through nitisinone, indicating correction of disease indicators in Fah-/- mice. CONCLUSIONS: The successful liver cell therapy for HT1 validates our protocol and, despite the known growth advantage of HT1, showcases ex vivo gene editing using electroporation in combination with liver cell therapy to cure a disease model. These advancements underscore the potential impacts of electroporation combined with transplantation as a cell therapy.


Subject(s)
Gene Editing , Hepatocytes , Hydrolases , Mice, Inbred C57BL , Tyrosinemias , Animals , Tyrosinemias/therapy , Tyrosinemias/genetics , Gene Editing/methods , Mice , Hepatocytes/transplantation , Hepatocytes/metabolism , Hydrolases/genetics , Cell- and Tissue-Based Therapy/methods , CRISPR-Cas Systems , Electroporation/methods , Mice, Knockout , 4-Hydroxyphenylpyruvate Dioxygenase/genetics , Disease Models, Animal , Cyclohexanones , Nitrobenzoates
10.
Microbiol Spectr ; 12(4): e0403523, 2024 Apr 02.
Article in English | MEDLINE | ID: mdl-38466097

ABSTRACT

With almost a quadrillion individuals, the Antarctic krill processes five million tons of organic carbon every day during austral summer. This high carbon flux requires a broad range of hydrolytic enzymes to decompose the diverse food-derived biopolymers. While krill itself possesses numerous such enzymes, it is unclear, to what extent the endogenous microbiota contribute to the hydrolytic potential of the gut environment. Here we applied amplicon sequencing, shotgun metagenomics, cultivation, and physiological assays to characterize the krill gut microbiota. The broad bacterial diversity (273 families, 919 genera, and 2,309 species) also included a complex potentially anaerobic sub-community. Plate-based assays with 198 isolated pure cultures revealed widespread capacities to utilize lipids (e.g., tributyrin), followed by proteins (casein) and to a lesser extent by polysaccharides (e.g., alginate and chitin). While most isolates affiliated with the genera Pseudoalteromonas and Psychrobacter, also Rubritalea spp. (Verrucomicrobia) were observed. The krill gut microbiota growing on marine broth agar plates possess 13,012 predicted hydrolyses; 15-fold more than previously predicted from a transcriptome-proteome compendium of krill. Cultivation-independent and -dependent approaches indicated members of the families Flavobacteriaceae and Pseudoalteromonadaceae to dominate the capacities for lipid/protein hydrolysis and to provide a plethora of carbohydrate-active enzymes, sulfatases, and laminarin- or porphyrin-depolymerizing hydrolases. Notably, also the potential to hydrolyze plastics such as polyethylene terephthalate and polylactatide was observed, affiliating mostly with Moraxellaceae. Overall, this study shows extensive microbial diversity in the krill gut, and suggests that the microbiota likely play a significant role in the nutrient acquisition of the krill by enriching its hydrolytic enzyme repertoire.IMPORTANCEThe Antarctic krill (Euphausia superba) is a keystone species of the Antarctic marine food web, connecting the productivity of phyto- and zooplankton with the nutrition of the higher trophic levels. Accordingly, krill significantly contributes to biomass turnover, requiring the decomposition of seasonally varying plankton-derived biopolymers. This study highlights the likely role of the krill gut microbiota in this ecosystem function by revealing the great number of diverse hydrolases that microbes contribute to the krill gut environment. The here resolved repertoire of hydrolytic enzymes could contribute to the overall nutritional resilience of krill and to the general organic matter cycling under changing environmental conditions in the Antarctic sea water. Furthermore, the krill gut microbiome could serve as a valuable resource of cold-adapted hydrolytic enzymes for diverse biotechnological applications.


Subject(s)
Euphausiacea , Humans , Animals , Euphausiacea/metabolism , Ecosystem , Seasons , Hydrolases/genetics , Hydrolases/metabolism , Biopolymers/metabolism
11.
Elife ; 122024 Mar 15.
Article in English | MEDLINE | ID: mdl-38488661

ABSTRACT

R-loops are non-canonical DNA structures that form during transcription and play diverse roles in various physiological processes. Disruption of R-loop homeostasis can lead to genomic instability and replication impairment, contributing to several human diseases, including cancer. Although the molecular mechanisms that protect cells against such events are not fully understood, recent research has identified fork protection factors and DNA damage response proteins as regulators of R-loop dynamics. In this study, we identify the Werner helicase-interacting protein 1 (WRNIP1) as a novel factor that counteracts transcription-associated DNA damage upon replication perturbation. Loss of WRNIP1 leads to R-loop accumulation, resulting in collisions between the replisome and transcription machinery. We observe co-localization of WRNIP1 with transcription/replication complexes and R-loops after replication perturbation, suggesting its involvement in resolving transcription-replication conflicts. Moreover, WRNIP1-deficient cells show impaired replication restart from transcription-induced fork stalling. Notably, transcription inhibition and RNase H1 overexpression rescue all the defects caused by loss of WRNIP1. Importantly, our findings highlight the critical role of WRNIP1 ubiquitin-binding zinc finger (UBZ) domain in preventing pathological persistence of R-loops and limiting DNA damage, thereby safeguarding genome integrity.


Subject(s)
ATPases Associated with Diverse Cellular Activities , DNA Replication , DNA-Binding Proteins , Humans , ATPases Associated with Diverse Cellular Activities/metabolism , DNA , DNA Damage , DNA-Binding Proteins/metabolism , Genomic Instability , Hydrolases/genetics , Zinc Fingers
12.
Science ; 383(6689): 1318-1325, 2024 Mar 22.
Article in English | MEDLINE | ID: mdl-38513014

ABSTRACT

Plants are constantly exposed to volatile organic compounds (VOCs) that are released during plant-plant communication, within-plant self-signaling, and plant-microbe interactions. Therefore, understanding VOC perception and downstream signaling is vital for unraveling the mechanisms behind information exchange in plants, which remain largely unexplored. Using the hormone-like function of volatile terpenoids in reproductive organ development as a system with a visual marker for communication, we demonstrate that a petunia karrikin-insensitive receptor, PhKAI2ia, stereospecifically perceives the (-)-germacrene D signal, triggering a KAI2-mediated signaling cascade and affecting plant fitness. This study uncovers the role(s) of the intermediate clade of KAI2 receptors, illuminates the involvement of a KAI2ia-dependent signaling pathway in volatile communication, and provides new insights into plant olfaction and the long-standing question about the nature of potential endogenous KAI2 ligand(s).


Subject(s)
Furans , Hydrolases , Petunia , Pyrans , Volatile Organic Compounds , Hydrolases/genetics , Hydrolases/metabolism , Signal Transduction , Volatile Organic Compounds/metabolism , Petunia/physiology , Furans/metabolism , Pyrans/metabolism , Sesquiterpenes, Germacrane/metabolism
13.
Gene ; 913: 148354, 2024 Jun 30.
Article in English | MEDLINE | ID: mdl-38492611

ABSTRACT

BACKGROUND: There are four distinct forms of Sanfilippo syndrome (MPS type III), each of which is an autosomal lysosomal storage disorder. These forms are caused by abnormalities in one of four lysosomal enzymes. This study aimed to identify possible genetic variants that contribute to Sanfilippo IIIB in 14 independent families in Southwest Iran. METHODS: Patients were included if their clinical features and enzyme assay results were suggestive. The patients were subsequently subjected to Sanger Sequencing to screen for Sanfilippo-related genes. Additional investigations have been conducted using various computational analyses to determine the probable functional effects of diagnosed variants. RESULTS: Five distinct variations were identified in the NAGLU gene. This included two novel variants in two distinct families and three previously reported variants in 12 distinct families. All of these variations were recognized as pathogenic using the MutationTaster web server. In silico analysis showed that all detected variants affected protein structural stability; four destabilized protein structures, and the fifth variation had the opposite effect. CONCLUSION: In this study, two novel variations in the NAGLU gene were identified. The results of this study positively contribute to the mutation diversity of the NAGLU gene. To identify new disease biomarkers and therapeutic targets, precision medicine must precisely characterize and account for genetic variations. New harmful gene variants are valuable for updating gene databases concerning Sanfilippo disease variations and NGS gene panels. This may also improve genetic counselling for rapid risk examinations and disease surveillance.


Subject(s)
Mucopolysaccharidosis III , Humans , Mucopolysaccharidosis III/genetics , Acetylglucosaminidase/genetics , Mutation , Hydrolases/genetics , Genetic Counseling
14.
Int J Biol Macromol ; 263(Pt 1): 130678, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38458276

ABSTRACT

Kiwifruit is a climacteric fruit that is prone to ripening and softening. Understanding molecular regulatory mechanism of kiwifruit softening, is helpful to ensure long-term storage of fruit. In the study, two NAC TFs and two XTH genes were isolated from kiwifruit. Phylogenetic tree showed that both AcNAC1 and AcNAC2 belonged to NAP subfamily, AcXTH1 belong to I subfamily, and AcXTH2 belong to III subfamily. Bioinformatics analysis predicted that AcNAC1 and AcNAC2 possessed similar three-dimensional structural, and belonged to hydrophilic proteins. AcXTH1 and AcXTH2 were hydrophilic proteins and contained signal peptides. AcXTH1 had a transmembrane structure, but AcXTH2 did not. qRT-PCR results showed that AcNAC1, AcNAC2, AcXTH1 and AcXTH2 were increased during kiwifruit ripening. Correlation analysis showed that kiwifruit softening was closely related to endotransglucosylase/hydrolase genes and NAC TFs, as well as there was also a close relationship between AcXTHs and AcNACs. Moreover, both AcNAC1 and AcNAC2 were transcriptional activators located in nucleus, which bound to and activated the promoters of AcXTH1 and AcXTH2. In shortly, we proved that the roles of NAC TFs in mediating fruit softening during kiwifruit ripening. Altogether, our results clarified that AcNAC1 and AcNAC2 were transcriptional activators, and took part in kiwifruit ripening and softening through activating endotransglucosylase/hydrolase genes, providing a new insight of fruit softening network in kiwifruit ripening.


Subject(s)
Actinidia , Fruit , Glycosyltransferases , Fruit/genetics , Fruit/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Phylogeny , Plant Proteins/genetics , Plant Proteins/metabolism , Actinidia/genetics , Actinidia/metabolism , Hydrolases/genetics , Gene Expression Regulation, Plant
15.
Microbiol Spectr ; 12(4): e0144523, 2024 Apr 02.
Article in English | MEDLINE | ID: mdl-38411054

ABSTRACT

Arginine catabolism by the bacterial arginine deiminase system (ADS) has anticariogenic properties through the production of ammonia, which modulates the pH of the oral environment. Given the potential protective capacity of the ADS pathway, the exploitation of ADS-competent oral microbes through pre- or probiotic applications is a promising therapeutic target to prevent tooth decay. To date, most investigations of the ADS in the oral cavity and its relation to caries have focused on indirect measures of activity or on specific bacterial groups, yet the pervasiveness and rate of expression of the ADS operon in diverse mixed microbial communities in oral health and disease remain an open question. Here, we use a multivariate approach, combining ultra-deep metatranscriptomic sequencing with paired metataxonomic and in vitro citrulline quantification to characterize the microbial community and ADS operon expression in healthy and late-stage cavitated teeth. While ADS activity is higher in healthy teeth, we identify multiple bacterial lineages with upregulated ADS activity on cavitated teeth that are distinct from those found on healthy teeth using both reference-based mapping and de novo assembly methods. Our dual metataxonomic and metatranscriptomic approach demonstrates the importance of species abundance for gene expression data interpretation and that patterns of differential expression can be skewed by low-abundance groups. Finally, we identify several potential candidate probiotic bacterial lineages within species that may be useful therapeutic targets for the prevention of tooth decay and propose that the development of a strain-specific, mixed-microbial probiotic may be a beneficial approach given the heterogeneity of taxa identified here across health groups. IMPORTANCE: Tooth decay is the most common preventable chronic disease, affecting more than two billion people globally. The development of caries on teeth is primarily a consequence of acid production by cariogenic bacteria that inhabit the plaque microbiome. Other bacterial strains in the oral cavity may suppress or prevent tooth decay by producing ammonia as a byproduct of the arginine deiminase metabolic pathway, increasing the pH of the plaque biofilm. While the benefits of arginine metabolism on oral health have been extensively documented in specific bacterial groups, the prevalence and consistency of arginine deiminase system (ADS) activity among oral bacteria in a community context remain an open question. In the current study, we use a multi-omics approach to document the pervasiveness of the expression of the ADS operon in both health and disease to better understand the conditions in which ADS activity may prevent tooth decay.


Subject(s)
Dental Caries , Microbiota , Humans , Ammonia/metabolism , Hydrolases/genetics , Hydrolases/metabolism , Microbiota/genetics , Arginine/metabolism
16.
Proc Natl Acad Sci U S A ; 121(10): e2312652121, 2024 Mar 05.
Article in English | MEDLINE | ID: mdl-38408229

ABSTRACT

Metformin is the first-line treatment for type II diabetes patients and a pervasive pollutant with more than 180 million kg ingested globally and entering wastewater. The drug's direct mode of action is currently unknown but is linked to effects on gut microbiomes and may involve specific gut microbial reactions to the drug. In wastewater treatment plants, metformin is known to be transformed by microbes to guanylurea, although genes encoding this metabolism had not been elucidated. In the present study, we revealed the function of two genes responsible for metformin decomposition (mfmA and mfmB) found in isolated bacteria from activated sludge. MfmA and MfmB form an active heterocomplex (MfmAB) and are members of the ureohydrolase protein superfamily with binuclear metal-dependent activity. MfmAB is nickel-dependent and catalyzes the hydrolysis of metformin to dimethylamine and guanylurea with a catalytic efficiency (kcat/KM) of 9.6 × 103 M-1s-1 and KM for metformin of 0.82 mM. MfmAB shows preferential activity for metformin, being able to discriminate other close substrates by several orders of magnitude. Crystal structures of MfmAB show coordination of binuclear nickel bound in the active site of the MfmA subunit but not MfmB subunits, indicating that MfmA is the active site for the MfmAB complex. Mutagenesis of residues conserved in the MfmA active site revealed those critical to metformin hydrolase activity and its small substrate binding pocket allowed for modeling of bound metformin. This study characterizes the products of the mfmAB genes identified in wastewater treatment plants on three continents, suggesting that metformin hydrolase is widespread globally in wastewater.


Subject(s)
Diabetes Mellitus, Type 2 , Guanidine/analogs & derivatives , Metformin , Microbiota , Urea/analogs & derivatives , Humans , Metformin/metabolism , Wastewater , Nickel , Hydrolases/genetics , Pharmaceutical Preparations
17.
J Agric Food Chem ; 72(6): 3025-3035, 2024 Feb 14.
Article in English | MEDLINE | ID: mdl-38300990

ABSTRACT

Zearalenone (ZEN) is a widespread mycotoxin that causes serious damage to animal husbandry and poses a threat to human health. A screen of ZEN-degrading soil bacteria yielded Bacillus subtilis YT-4, which yielded 80% ZEN degradation after 6 h and 95% after 36 h. The gene sequence encoding the degradative enzyme ZENY was mined from the genome of YT-4 and expressed in yeast. ZENY is an α/ß-hydrolase with an optimal enzyme activity at 37 °C and pH 8. By breaking the lactone ring of ZEN, it produces ZENY-C18H24O5 with a molecular weight of 320.16 g/mol. Sequence comparison and molecular docking analyses identified the catalytic ZENY triad 99S-245H-123E and the primary ZEN-binding mode within the hydrophobic pocket of the enzyme. To improve the thermal stability of the enzyme for industrial applications, we introduced a mutation at the N-terminus, specifically replacing the fifth residue N with V, and achieved a 25% improvement in stability at 45 °C. These findings aim to achieve ZEN biodegradation and provide insight into the structure and function of ZEN hydrolases.


Subject(s)
Zearalenone , Animals , Humans , Zearalenone/metabolism , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Molecular Docking Simulation , Hydrolases/genetics , Mutation
18.
Mol Biol Rep ; 51(1): 327, 2024 Feb 23.
Article in English | MEDLINE | ID: mdl-38393446

ABSTRACT

BACKGROUND: In the present study, we examine the prevalence of phylogenetic groups, O-serogroups, adhesin genes, antimicrobial resistance, the level of gene expression associated with biofilm formation, and the presence of extended-spectrum beta-lactamase (ESBL) in UPEC strains isolated from both pediatric and adult patients. METHODS: In this cross-sectional study, 156 UPEC isolates were collected from UTI patients. ESBL-producing isolates were detected using the double-disc synergy (DDS) method, and biofilm formation was assessed through a microplate assay. The presence of O-serogroups, adhesion factors and resistance genes, including ESBLs and PMQR genes, was detected by PCR, and isolates were categorized into phylogenetic groups using multiplex PCR. Additionally, the quantitative real-time PCR method was also used to determine the expression level of genes related to biofilm. RESULTS: During the study period, 50.6% (79/156) of the samples were obtained from children, and 49.4% (77/156) were from adults. The highest rate of resistance was to NA (91.7%), while FM (10.9%) had the lowest rate of antibiotic resistance. In addition, 67.9% (106/156) of UPEC isolates were ESBL producers. Most of UPEC isolates belonged to phylogenetic group B2 (37.1%). This study revealed that blaCTX-M and qnrS are widely distributed among UPEC isolates. The mean expression levels of fimA genes were significantly higher in non-biofilm producers than in biofilm producers (p < 0.01). CONCLUSIONS: The high antibiotic resistance rates in this study highlight the significance of local resistance monitoring and investigating underlying mechanisms. Our findings indicate the dominance of phylogroup B2 and group D as the prevailing phylogenetic groups. Consequently, it is imperative to investigate the epidemiological aspects and characterize UPEC isolates across diverse regions and time frames.


Subject(s)
Escherichia coli Infections , Urinary Tract Infections , Uropathogenic Escherichia coli , Adult , Humans , Child , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Phylogeny , Uropathogenic Escherichia coli/genetics , Escherichia coli Infections/genetics , Escherichia coli Infections/drug therapy , Cross-Sectional Studies , Drug Resistance, Bacterial/genetics , Hydrolases/genetics , Biofilms , Urinary Tract Infections/drug therapy
19.
J Biol Chem ; 300(3): 105783, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38395309

ABSTRACT

Poly(ethylene terephthalate) (PET) is a major plastic polymer utilized in the single-use and textile industries. The discovery of PET-degrading enzymes (PETases) has led to an increased interest in the biological recycling of PET in addition to mechanical recycling. IsPETase from Ideonella sakaiensis is a candidate catalyst, but little is understood about its structure-function relationships with regards to PET degradation. To understand the effects of mutations on IsPETase productivity, we develop a directed evolution assay to identify mutations beneficial to PET film degradation at 30 °C. IsPETase also displays enzyme concentration-dependent inhibition effects, and surface crowding has been proposed as a causal phenomenon. Based on total internal reflectance fluorescence microscopy and adsorption experiments, IsPETase is likely experiencing crowded conditions on PET films. Molecular dynamics simulations of IsPETase variants reveal a decrease in active site flexibility in free enzymes and reduced probability of productive active site formation in substrate-bound enzymes under crowding. Hence, we develop a surface crowding model to analyze the biochemical effects of three hit mutations (T116P, S238N, S290P) that enhanced ambient temperature activity and/or thermostability. We find that T116P decreases susceptibility to crowding, resulting in higher PET degradation product accumulation despite no change in intrinsic catalytic rate. In conclusion, we show that a macromolecular crowding-based biochemical model can be used to analyze the effects of mutations on properties of PETases and that crowding behavior is a major property to be targeted for enzyme engineering for improved PET degradation.


Subject(s)
Burkholderiales , Hydrolases , Polyethylene Terephthalates , Hydrolases/chemistry , Hydrolases/genetics , Hydrolases/metabolism , Polyethylene Terephthalates/chemistry , Polyethylene Terephthalates/metabolism , Recycling , Kinetics , Burkholderiales/enzymology , Models, Chemical
20.
J Integr Neurosci ; 23(1): 16, 2024 Jan 16.
Article in English | MEDLINE | ID: mdl-38287861

ABSTRACT

BACKGROUND: Mutations in the glucocerebrosidase (GBA1) and leucine-rich repeat kinase 2 (LRRK2) genes, encoding lysosomal enzyme glucocerebrosidase (GCase) and leucine-rich repeat kinase 2 (LRRK2), respectively, are the most common related to Parkinson's disease (PD). Recent data suggest a possible functional interaction between GCase and LRRK2 and their involvement in sphingolipid metabolism. The aim of the present study was to describe the clinical course and evaluate the lysosomal enzyme activities and sphingolipid concentrations in blood of patients with PD associated with dual mutations p.N370S GBA1 and p.G2019S LRRK2 (p.N370S/GBA-p.G2019S/LRRK2-PD) as well as in blood of asymptomatic mutation carriers (p.N370S/GBA1-p.G2019S/LRRK2-carrier). METHODS: One patient with p.N370S/GBA1-p.G2019S/LRRK2-PD and one p.N370S/GBA1-p.G2019S/LRRK2-carrier were enrolled. GBA1-associated PD (GBA1-PD), LRRK2-associated PD (LRRK2-PD), sporadic PD (sPD) patients were described earlier by our research group. A neuropsychiatric examination of the p.N370S/GBA1-p.G2019S/LRRK2-PD patient was carried out using scales (Montreal Cognitive Assessment scale (MoCA), Mini-mental State Examination scale (MMSE), Frontal Assessment Batter scale (FAB), Hospital Anxiety, and Depression Scale (HADS), etc). Lysosomal enzyme activity (GCase, alpha-galactosidase [GLA], acid sphingomyelinase [ASMase], galactosylcerebrosidase [GALC]) and sphingolipid concentrations (hexasylsphingosine [HexSph], lysoglobotriaosylsphingosine [LysoGb3], lysosphingomyelin [LysoSM]) were assessed with high-performance liquid chromatography-tandem mass spectrometry in blood. The following comparison with the previously described groups of GBA1-PD and sPD patients were conducted. RESULTS: Clinical features of p.N370S/GBA1-p.G2019S/LRRK2-PD included an early age of onset of the disease (46 years) and mild cognitive and affective disorders (MMSE = 29, MoCA = 23), despite a long (24 years) course of the disease. Interestingly, no differences were found in hydrolase activity and lysosphingolipid concentrations between the p.N370S/GBA1-p.G2019S/LRRK2-PD patient and GBA1-PD patients. However, GCase activity was lower in these groups than in LRRK2-PD, sPD, and controls. Additionally, the p.N370S/GBA1-p.G2019S/LRRK2-PD patient was characterized by a pronounced decreased in ASMase activity and increased LysoSM concentration compared to the p.N370S/GBA1-p.G2019S/LRRK2-carrier (p = 0.023, p = 0.027, respectively). CONCLUSIONS: Based on one patient, our results indicate a protective effect of the p.G2019S mutation in the LRRK2 gene on clinical course of p.N370S/GBA1-PD. The identified pronounced alteration of ASMase activity and LysoSM concentration in p.N370S/GBA1-p.G2019S/LRRK2-PD provide the basis for the further research.


Subject(s)
Glucosylceramidase , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Parkinson Disease , Humans , Middle Aged , Disease Progression , Glucosylceramidase/genetics , Glucosylceramidase/metabolism , Hydrolases/genetics , Leucine/genetics , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Lysosomes/metabolism , Mutation , Parkinson Disease/genetics , Sphingolipids
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