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In various life forms, fucose-containing glycans play vital roles in immune recognition, developmental processes, plant immunity, and host-microbe interactions. Together with glucose, galactose, N -acetylglucosamine, and sialic acid, fucose is a significant component of human milk oligosaccharides (HMOs). Fucosylated HMOs benefit infants by acting as prebiotics, preventing pathogen attachment, and potentially protecting against infections, including HIV. Although the need for fucosylated derivatives is clear, their availability is limited. Therefore, synthesis methods for various fucosylated oligosaccharides are explored, employing enzymatic approaches and α-L-fucosidases. This work aimed to characterise α-L-fucosidases identified in an alpaca faeces metagenome. Based on bioinformatic analyses, they were confirmed as members of the GH29A subfamily. The recombinant α-L-fucosidases were expressed in Escherichia coli and showed hydrolytic activity towards p -nitrophenyl-α-L-fucopyranoside and 2'-fucosyllactose. Furthermore, the enzymes' biochemical properties and kinetic characteristics were also determined. All four α-L-fucosidases could catalyse transfucosylation using a broad diversity of fucosyl acceptor substrates, including lactose, maltotriose, L-serine, and L-threonine. The results contribute insights into the potential use of α-L-fucosidases for synthesising fucosylated amino acids.

Klebsiella pneumoniae poses a major global challenge due to its virulence, multidrug resistance, and nosocomial nature. Thus, bacteriophage-derived proteins are extensively being investigated as a means to combat this bacterium. In this study, we explored the enzymatic specificity of depolymerase gp531, encoded by the jumbo bacteriophage vB_KleM_RaK2 (RaK2). We used two different methods to modify the reducing end of the oligosaccharides released during capsule hydrolysis with gp531. Subsequent acidic cleavage with TFA, followed by TLC and HPLC-MS analyses, revealed that RaK2 gp531 is a β-(1→4)-endoglucosidase. The enzyme specifically recognizes and cleaves the capsular polysaccharide (CPS) of the Klebsiella pneumoniae K54 serotype, releasing K-unit monomers (the main product), dimers, and trimers. Depolymerase gp531 remains active from 10 to 50 °C and in the pH 3-8 range, indicating its stability and versatility. Additionally, we demonstrated that gp531's activity is not affected by CPS acetylation, which is influenced by the growth conditions of the bacterial culture. Overall, our findings provide valuable insights into the enzymatic activity of the first characterized depolymerase targeting the capsule of the clinically relevant K54 serotype of K. pneumoniae., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Paenibacillus sp. GKG was isolated based on its ability to produce hydrolysis zones on agar plates containing yeast cell wall substrate as the single carbon source. The extracellular enzymes secreted into the culture medium were identified by LC-MS/MS proteomics. Endo-β-1,3-glucanase PsLam81A containing GH81 catalytic and the CBM56 carbohydrate-binding modules was selected for heterologous expression in Escherichia coli . The identity of the recombinant PsLam81A was confirmed by LC-MS/MS proteomics. The PsLam81A showed the highest activity at 60 °C, and the optimal pH range was between 6.5 and 8.0. The analysis of the full-length PsLam81A and truncated PsLam81AΔCBM56 enzymes showed that the CBM56 module improved the hydrolytic activity towards linear β-1,3-glucans-curdlan and pachyman but had no effect on hydrolysis of β-1,3/β1,6-branched glucans-laminarin and yeast β-glucan. The characterization of PsLam81A enzyme broadens current knowledge on the biochemical properties and substrate specificity of family 81 glycoside hydrolases and allows prediction of the necessity of CBM56 module in the process of designing new truncated or chimeric glycosidases.

We recently discovered a [Fe-S]-containing protein with in vivo thiouracil desulfidase activity, dubbed TudS. The crystal structure of TudS refined at 1.5 Å resolution is reported; it harbors a [4Fe-4S] cluster bound by three cysteines only. Incubation of TudS crystals with 4-thiouracil trapped the cluster with a hydrosulfide ligand bound to the fourth non-protein-bonded iron, as established by the sulfur anomalous signal. This indicates that a [4Fe-5S] state of the cluster is a catalytic intermediate in the desulfuration reaction. Structural data and site-directed mutagenesis indicate that a water molecule is located next to the hydrosulfide ligand and to two catalytically important residues, Ser101 and Glu45. This information, together with modeling studies allow us to propose a mechanism for the unprecedented non-redox enzymatic desulfuration of thiouracil, in which a [4Fe-4S] cluster binds and activates the sulfur atom of the substrate., (© 2020 Wiley-VCH GmbH.)

To maximize the potential of 5G infrastructure in healthcare, simple integration of biosensors with wireless tag antennas would be beneficial. This work introduces novel glucose-to-resistor transduction, which enables simple, wireless biosensor design. The biosensor was realized on a near-field communication tag antenna, where a sensing bioanode generated electrical current and electroreduced a nonconducting antenna material into an excellent conductor. For this, a part of the antenna was replaced by a Ag nanoparticle layer oxidized to high-resistance AgCl. The bioanode was based on Au nanoparticle-wired glucose dehydrogenase (GDH). The exposure of the cathode-bioanode to glucose solution resulted in GDH-catalyzed oxidation of glucose at the bioanode with a concomitant reduction of AgCl to highly conducting Ag on the cathode. The AgCl-to-Ag conversion strongly affected the impedance of the antenna circuit, allowing wireless detection of glucose. Mimicking the final application, the proposed wireless biosensor was ultimately evaluated through the measurement of glucose in whole blood, showing good agreement with the values obtained with a commercially available glucometer. This work, for the first time, demonstrates that making a part of the antenna from the AgCl layer allows achieving simple, chip-less, and battery-less wireless sensing of enzyme-catalyzed reduction reaction.

Shiga toxin-producing Escherichia coli (STEC) O26:H11 is an emerging foodborne pathogen of growing concern. Since current strategies to control microbial contamination in foodstuffs do not guarantee the elimination of O26:H11, novel approaches are needed. Bacteriophages present an alternative to traditional biocontrol methods used in the food industry. Here, a previously isolated bacteriophage vB&#95;EcoM&#95;VR26 (VR26), adapted to grow at common refrigeration temperatures (4 and 8 °C), has been evaluated for its potential as a biocontrol agent against O26:H11. After 2 h of treatment in broth, VR26 reduced O26:H11 numbers ( p < 0.01) by > 2 log 10 at 22 °C, and ~3 log 10 at 4 °C. No bacterial regrowth was observed after 24 h of treatment at both temperatures. When VR26 was introduced to O26:H11-inoculated lettuce, ~2.0 log 10 CFU/piece reduction was observed at 4, 8, and 22 °C. No survivors were detected after 4 and 6 h at 8 and 4 °C, respectively. Although at 22 °C, bacterial regrowth was observed after 6 h of treatment, O26:H11 counts on non-treated samples were >2 log 10 CFU/piece higher than on phage-treated ones ( p < 0.02). This, and the ability of VR26 to survive over a pH range of 3-11, indicates that VR26 could be used to control STEC O26:H11 in the food industry.

Parvovirus B19 (B19V) is a widespread human pathogen possessing a high tropism for erythroid precursor cells. However, the persistence or active replication of B19V in endothelial cells (EC) has been detected in diverse human pathologies. The VP1 unique region (VP1u) of the viral capsid has been reported to act as a major determinant of viral tropism for erythroid precursor cells. Nevertheless, the interaction of VP1u with EC has not been studied. We demonstrate that recombinant VP1u is efficiently internalized by rats' pulmonary trunk blood vessel-derived EC in vitro compared to the human umbilical vein EC line. The exposure to VP1u was not acutely cytotoxic to either human- or rat-derived ECs, but led to the upregulation of cellular stress signaling-related pathways. Our data suggest that high levels of circulating B19V during acute infection can cause endothelial damage, even without active replication or direct internalization into the cells.

Pyridine and its derivatives constitute the majority of heterocyclic aromatic compounds that occur largely as a result of human activities and contribute to environmental pollution. It is known that they can be degraded by various bacteria in the environment; however, the degradation of unsubstituted pyridine has not yet been completely resolved. In this study, we present data on the pyridine catabolic pathway in Arthrobacter sp. strain 68b at the level of genes, enzymes, and metabolites. The pyr gene cluster, responsible for the degradation of pyridine, was identified in a catabolic plasmid, p2MP. The pathway of pyridine metabolism consisted of four enzymatic steps and ended by the formation of succinic acid. The first step in the degradation of pyridine proceeds through a direct ring cleavage catalyzed by a two-component flavin-dependent monooxygenase system, encoded by pyrA (pyridine monooxygenase) and pyrE genes. The genes pyrB , pyrC , and pyrD were found to encode ( Z )- N -(4-oxobut-1-enyl)formamide dehydrogenase, amidohydrolase, and succinate semialdehyde dehydrogenase, respectively. These enzymes participate in the subsequent steps of pyridine degradation. The metabolites of these enzymatic reactions were identified, and this allowed us to reconstruct the entire pyridine catabolism pathway in Arthrobacter sp. 68b. IMPORTANCE The biodegradation pathway of pyridine, a notorious toxicant, is relatively unexplored, as no genetic data related to this process have ever been presented. In this paper, we describe the plasmid-borne pyr gene cluster, which includes the complete set of genes responsible for the degradation of pyridine. A key enzyme, the monooxygenase PyrA, which is responsible for the first step of the catabolic pathway, performs an oxidative cleavage of the pyridine ring without typical activation steps such as reduction or hydroxylation of the heterocycle. This work provides new insights into the metabolism of N -heterocyclic compounds in nature., (Copyright © 2020 American Society for Microbiology.)

Human activating signal cointegrator homology (ASCH) domain-containing proteins are widespread and diverse but, at present, the vast majority of those proteins have no function assigned to them. This study demonstrates that the 103-amino acid Escherichia coli protein YqfB, previously identified as hypothetical, is a unique ASCH domain-containing amidohydrolase responsible for the catabolism of N 4 -acetylcytidine (ac4C). YqfB has several interesting and unique features: i) it is the smallest monomeric amidohydrolase described to date, ii) it is active towards structurally different N 4 -acylated cytosines/cytidines, and iii) it has a high specificity for these substrates (k cat /K m up to 2.8 × 10 6  M -1  s -1 ). Moreover, our results suggest that YqfB contains a unique Thr-Lys-Glu catalytic triad, and Arg acting as an oxyanion hole. The mutant lacking the yqfB gene retains the ability to grow, albeit poorly, on N 4 -acetylcytosine as a source of uracil, suggesting that an alternative route for the utilization of this compound exists in E. coli. Overall, YqfB ability to hydrolyse various N 4 -acylated cytosines and cytidines not only sheds light on the long-standing mystery of how ac4C is catabolized in bacteria, but also expands our knowledge of the structural diversity within the active sites of amidohydrolases.

The recombinant phage tail sheath protein, gp053, from Escherichia coli infecting myovirus vB&#95;EcoM&#95;FV3 (FV3) was able to self-assemble into long, ordered and extremely stable tubular structures (polysheaths) in the absence of other viral proteins. TEM observations revealed that those protein nanotubes varied in length (~10⁻1000 nm). Meanwhile, the width of the polysheaths (~28 nm) corresponded to the width of the contracted tail sheath of phage FV3. The formed protein nanotubes could withstand various extreme treatments including heating up to 100 °C and high concentrations of urea. To determine the shortest variant of gp053 capable of forming protein nanotubes, a set of N- or/and C-truncated as well as poly-His-tagged variants of gp053 were constructed. The TEM analysis of these mutants showed that up to 25 and 100 amino acid residues could be removed from the N and C termini, respectively, without disturbing the process of self-assembly. In addition, two to six copies of the gp053 encoding gene were fused into one open reading frame. All the constructed oligomers of gp053 self-assembled in vitro forming structures of different regularity. By using the modification of cysteines with biotin, the polysheaths were tested for exposed thiol groups. Polysheaths formed by the wild-type gp053 or its mutants possess physicochemical properties, which are very attractive for the construction of self-assembling nanostructures with potential applications in different fields of nanosciences.

Ribose methylation is among the most ubiquitous modifications found in RNA. 2'- O -methyluridine is found in rRNA, snRNA, snoRNA and tRNA of Archaea , Bacteria , and Eukaryota . Moreover, 2'- O -methylribonucleosides are promising starting materials for the production of nucleic acid-based drugs. Despite the countless possibilities of practical use for the metabolic enzymes associated with methylated nucleosides, there are very few reports regarding the metabolic fate and enzymes involved in the metabolism of 2'- O -alkyl nucleosides. The presented work focuses on the cellular degradation of 2'- O -methyluridine. A novel enzyme was found using a screening strategy that employs Escherichia coli uracil auxotroph and the metagenomic libraries. A 2'- O -methyluridine hydrolase (RK9NH) has been identified together with an aldolase (RK9DPA)-forming a part of a probable gene cluster that is involved in the degradation of 2'- O -methylated nucleosides. The RK9NH is functional in E. coli uracil auxotroph and in vitro. The RK9NH nucleoside hydrolase could be engineered to enzymatically produce 2'- O -methylated nucleosides that are of great demand as raw materials for production of nucleic acid-based drugs. Moreover, RK9NH nucleoside hydrolase converts 5-fluorouridine, 5-fluoro-2'-deoxyuridine and 5-fluoro-2'- O -methyluridine into 5-fluorouracil, which suggests it could be employed in cancer therapy.

Cytosine is one of the four letters of a standard genetic code, found both in DNA and in RNA. This heterocyclic base can be converted into uracil upon the action of the well-known cytosine deaminase. Isocytosine (2-aminouracil) is an isomer of cytosine, yet the enzymes that could convert it into uracil were previously mainly overlooked. In order to search for the isocytosine deaminases we used a selection strategy that is based on uracil auxotrophy and the metagenomic libraries, which provide a random pool of genes from uncultivated soil bacteria. Several genes that encode isocytosine deaminases were found and two respective recombinant proteins were purified. It was established that both novel deaminases do not use cytosine as a substrate. Instead, these enzymes are able to convert not only isocytosine into uracil, but also 5-fluoroisocytosine into 5-fluorouracil. Our findings suggest that novel isocytosine deaminases have a potential to be efficiently used in targeted cancer therapy instead of the classical cytosine deaminases. Use of isocytosine instead of cytosine would produce fewer side effects since deaminases produced by the commensal E. coli gut flora are ten times less efficient in degrading isocytosine than cytosine. In addition, there are no known homologs of isocytosine deaminases in human cells that would induce the toxicity when 5-fluoroisocytosine would be used as a prodrug.

Modified nucleotides are present in many RNA species in all Domains of Life. While the biosynthetic pathways of such nucleotides are well studied, much less is known about the degradation of RNAs and the return to the metabolism of modified nucleotides, their respective nucleosides or heterocyclic bases. Using an E. coli uracil auxotroph, we screened the metagenomic libraries for genes, which would allow the conversion of 2-thiouracil to uracil and thereby lead to the growth on a defined synthetic medium. We show that a gene encoding a protein consisting of previously uncharacterized Domain of Unknown Function 523 (DUF523) is responsible for such phenotype. We have purified this recombinant protein and demonstrated that it contains a FeS cluster. The substitution of cysteines, which have been predicted to form such clusters, with alanines abolished the growth phenotype. We conclude that DUF523 is involved in the conversion of 2-thiouracil into uracil in vivo., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Tricyclic wyosine derivatives are found at position 37 of eukaryotic and archaeal tRNA Phe In Archaea, the intermediate imG-14 is targeted by three different enzymes that catalyze the formation of yW-86, imG, and imG2. We have suggested previously that a peculiar methyltransferase (aTrm5a/Taw22) likely catalyzes two distinct reactions: N 1 -methylation of guanosine to yield m 1 G; and C 7 -methylation of imG-14 to yield imG2. Here we show that the recombinant aTrm5a/Taw22-like enzymes from both Pyrococcus abyssi and Nanoarchaeum equitans indeed possess such dual specificity. We also show that substitutions of individual conservative amino acids of P. abyssi Taw22 (P260N, E173A, and R174A) have a differential effect on the formation of m 1 G/imG2, while replacement of R134, F165, E213, and P262 with alanine abolishes the formation of both derivatives of G37. We further demonstrate that aTrm5a-type enzyme SSO2439 from Sulfolobus solfataricus, which has no N 1 -methyltransferase activity, exhibits C 7 -methyltransferase activity, thereby producing imG2 from imG-14. We thus suggest renaming such aTrm5a methyltransferases as Taw21 to distinguish between monofunctional and bifunctional aTrm5a enzymes., (© 2016 Urbonavičius et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Rhodococcus rhodochrous PY11 (DSM 101666) is able to use 2-hydroxypyridine as a sole source of carbon and energy. By investigating a gene cluster (hpo) from this bacterium, we were able to reconstruct the catabolic pathway of 2-hydroxypyridine degradation. Here, we report that in Rhodococcus rhodochrous PY11, the initial hydroxylation of 2-hydroxypyridine is catalyzed by a four-component dioxygenase (HpoBCDF). A product of the dioxygenase reaction (3,6-dihydroxy-1,2,3,6-tetrahydropyridin-2-one) is further oxidized by HpoE to 2,3,6-trihydroxypyridine, which spontaneously forms a blue pigment. In addition, we show that the subsequent 2,3,6-trihydroxypyridine ring opening is catalyzed by the hypothetical cyclase HpoH. The final products of 2-hydroxypyridine degradation in Rhodococcus rhodochrous PY11 are ammonium ion and α-ketoglutarate., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background. Production of highly pure enantiomers of vicinal diols is desirable, but difficult to achieve. Enantiomerically pure diols and acyloins are valuable bulk chemicals, promising synthones and potential building blocks for chiral polymers. Enzymatic reduction of ketones is a useful technique for the synthesis of the desired enantiomeric alcohols. Here, we report on the characterization of a ketoreductase TpdE from Rhodococcus jostii TMP1 that is a prospective tool for the synthesis of such compounds. Results. In this study, NADPH-dependent short-chain dehydrogenase/reductase TpdE from Rhodococcus jostii TMP1 was characterized. The enzyme exhibited broad substrate specificity towards aliphatic 2,3-diketones, butan-3-one-2-yl alkanoates, as well as acetoin and its acylated derivatives. TpdE stereospecifically reduced α-diketones to the corresponding diols. The GC-MS analysis of the reduction products of 2,3- and 3,4-diketones indicated that TpdE is capable of reducing both keto groups in its substrate leading to the formation of two new chiral atoms in the product molecule. Bioconversions of diketones to corresponding diols occurred using either purified enzyme or a whole-cell Escherichia coli BL21 (DE3) biocatalyst harbouring recombinant TpdE. The optimum temperature and pH were determined to be 30-35 °C and 7.5, respectively. Conclusions. The broad substrate specificity and stereoselectivity of TpdE from Rhodococcus jostii TMP1 make it a promising biocatalyst for the production of enantiomerically pure diols that are difficult to obtain by chemical routes.

A range of diseases is associated with amyloid fibril formation. Despite different proteins being responsible for each disease, all of them share similar features including beta-sheet-rich secondary structure and fibril-like protein aggregates. A number of proteins can form amyloid-like fibrils in vitro, resembling structural features of disease-related amyloids. Given these generic structural properties of amyloid and amyloid-like fibrils, generic inhibitors of fibril formation would be of interest for treatment of amyloid diseases. Recently, we identified five outstanding inhibitors of insulin amyloid-like fibril formation among the pool of 265 commercially available flavone derivatives. Here we report testing of these five compounds and of epi-gallocatechine-3-gallate (EGCG) on aggregation of alpha-synuclein and beta-amyloid. We used a Thioflavin T (ThT) fluorescence assay, relying on halftimes of aggregation as the measure of inhibition. This method avoids large numbers of false positive results. Our data indicate that four of the five flavones and EGCG inhibit alpha-synuclein aggregation in a concentration-dependent manner. However none of these derivatives were able to increase halftimes of aggregation of beta-amyloid.

RNA modification has emerged as a crucial area of research in epigenetics, significantly influencing tumor biology by regulating RNA metabolism. N‐acetyltransferase 10 (NAT10)‐mediated N4‐acetylcytidine (ac4C) modification, the sole known acetylation in eukaryotic RNA, influences cancer pathogenesis and progression. NAT10 is the only writer of ac4C and catalyzes acetyl transfer on targeted RNA, and ac4C helps to improve the stability and translational efficiency of ac4C‐modified RNA. NAT10 is highly expressed and associated with poor prognosis in pan‐cancers. Based on its molecular mechanism and biological functions, ac4C is a central factor in tumorigenesis, tumor progression, drug resistance, and tumor immune escape. Despite the increasing focus on ac4C, the specific regulatory mechanisms of ac4C in cancer remain elusive. The present review thoroughly analyzes the current knowledge on NAT10‐mediated ac4C modification in cancer, highlighting its broad regulatory influence on targeted gene expression and tumor biology. This review also summarizes the limitations and perspectives of current research on NAT10 and ac4C in cancer, to identify new therapeutic targets and advance cancer treatment strategies. [ABSTRACT FROM AUTHOR]

Small angle X-ray diffraction (SAXD), resonance Raman (RR) spectroscopy with 413 nm excitation, and non-resonance Raman technique with 785 nm excitation were used to probe the influence of entrapped cytochrome c (Cyt c) on the structure of hydrated phytantriol (Phyt) liquid-crystalline phases as well as conformational changes of heme group and secondary structure of the protein. SAXD measurements indicated that incorporation of Cyt c affects both nanostructure dimensions and type of liquid-crystalline phases of hydrated Phyt. The unit cell dimensions decrease with increasing Cyt c concentration for all phases. In addition, protein perturbs the nanostructure of Q(230) and Q(224) liquid-crystalline phases of hydrated Phyt to such an extent that they transform into the Q(229) phase with the Im3m space group. RR data revealed that entrapment of oxidized Cyt c into the Q(230) phase at 1 wt.% content results in near complete reduction of central iron ion of the heme group, while its low-spin state and six-ligand coordination configuration are preserved. Based on the analysis of heme out-of-plane folding vibration near 568 cm(-1) (γ(21)) and ν(48) mode at 633 cm(-1), it was demonstrated that the protein matrix tension on the heme group is relaxed upon incorporation of protein into Q(230) phase. Non-resonant Raman bands of difference spectra showed the preservation of α-helix secondary structure of Cyt c in the liquid-crystalline phase at relatively high (5 wt.%) content. The Cyt c induced spectroscopic changes of Phyt bands were found to be similar as decrease in temperature., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Glycine betaine (GB) could be used by Arthrobacter globiformis cells as a sole carbon source. The cells took up this molecule in the low as well as in the high salinity medium. Addition of GB to the mineral medium with high salt concentration revealed that GB was also used as an osmoprotectant. Dimethylglycine oxidase (DMGO) was involved in the catabolism of GB. Two genes for DMGO were detected in a cloned 26267 bp fragment of A. globiformis DNA. The genes involved in the tetrahydrofolate-dependent assimilation of methyl groups were located nearby the two of DMGO genes. Both cloned A. globiformis DMGO were active. The activity of DMGO was detected in A. globiformis cells and it depended on the addition of GB and the salinity of the medium. Reverse transcription-PCR demonstrated that the addition of GB influenced the transcription of dmg genes.

The Apicomplexa phylum encompasses numerous obligate intracellular parasites, some associated with severe implications for human health, including Plasmodium, Cryptosporidium, and Toxoplasma gondii. The iron-sulfur cluster [Fe-S] biogenesis ISC pathway, localized within the mitochondrion or mitosome of these parasites, is vital for parasite survival and development. Previous work on T. gondii and Plasmodium falciparum provided insights into the mechanisms of [Fe-S] biogenesis within this phylum, while the transporter linking mitochondria-generated [Fe-S] with the cytosolic [Fe-S] assembly (CIA) pathway remained elusive. This critical step is catalyzed by a well-conserved ABC transporter, termed ATM1 in yeast, ATM3 in plants and ABCB7 in mammals. Here, we identify and characterize this transporter in two clinically relevant Apicomplexa. We demonstrate that depletion of TgATM1 does not specifically impair mitochondrial metabolism. Instead, proteomic analyses reveal that TgATM1 expression levels inversely correlate with the abundance of proteins that participate in the transfer of [Fe-S] to cytosolic proteins at the outer mitochondrial membrane. Further insights into the role of TgATM1 are gained through functional complementation with the well-characterized yeast homolog. Biochemical characterization of PfATM1 confirms its role as a functional ABC transporter, modulated by oxidized glutathione (GSSG) and [4Fe-4S]. Author summary: The Apicomplexa phylum includes harmful parasites like Plasmodium falciparum and Toxoplasma gondii, posing significant threats to human health. Understanding their survival mechanisms is crucial for developing effective treatments. Here, our focus is on iron-sulfur cluster [Fe-S] synthesis pathways found in the mitochondrion and cytosol, essential for the function of many enzymes. Although the transporter responsible for moving sulfur-containing compounds from mitochondria to the cytosol is known in other organisms, it remains unidentified in apicomplexans. Our study identifies PfATM1 and TgATM1 as critical transporters in the mitochondrial membrane of these parasites. Downregulation or overexpression of TgATM1 levels do not hinder mitochondrial functions but affect the level of other proteins, suspected to donate [Fe-S] to cytosolic proteins. We further confirm the transporter's role by rescuing TgATM1 depleted parasites with the yeast homolog ScATM1. Additionally, we identify glutathione-coordinated [4Fe-4S] as a probable substrate for PfATM1. These findings provide valuable insights into how parasites manage essential resources, potentially opening new avenues for targeted drug development. [ABSTRACT FROM AUTHOR]

In recent years, there has been increasing interest in studying gut microbiomederived hydrolases in relation to oral drug metabolism, particularly focusing on natural product drugs. Despite the significance of natural product drugs in the field of oral medications, there is a lack of research on the regulatory interplay between gut microbiome-derived hydrolases and these drugs. This review delves into the interaction between intestinal microbiome-derived hydrolases and natural product drugs metabolism from three key perspectives. Firstly, it examines the impact of glycoside hydrolases, amide hydrolases, carboxylesterase, bile salt hydrolases, and epoxide hydrolase on the structure of natural products. Secondly, it explores how natural product drugs influence microbiome-derived hydrolases. Lastly, it analyzes the impact of interactions between hydrolases and natural products on disease development and the challenges in developing microbial-derived enzymes. The overarching goal of this review is to lay a solid theoretical foundation for the advancement of research and development in new natural product drugs and personalized treatment. [ABSTRACT FROM AUTHOR]

Prion diseases such as scrapie, bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD) affect domesticated and wild herbivorous mammals. Animals afflicted with CWD, the transmissible spongiform encephalopathy of cervids (deer, elk, and moose), shed prions into the environment, where they may persist and remain infectious for years. These environmental prions may remain in soil, be transported in surface waters, or assimilated into plants. Environmental sampling is an emerging area of TSE research and can provide more information about prion fate and transport once shed by infected animals. In this study, we have developed the first published method for the extraction and detection of prions in plant tissue using the real-time quaking-induced conversion (RT-QuIC) assay. Incubation with a zwitterionic surfactant followed by precipitation with sodium phosphotungstate concentrates the prions within samples and allows for sensitive detection of prion seeding activity. Using this protocol, we demonstrate that prions can be detected within plant tissues and on plant surfaces using the RT-QuIC assay. [ABSTRACT FROM AUTHOR]

High-fiber, low-cost agricultural byproducts offer a sustainable alternative for mitigating the competition for crops between humans and livestock. Pigs predominantly utilize dietary fibers through the process of microbial fermentation within the gut. This study explored the gut microbiota and the capacity for carbohydrate degradation in 30 individual Lantang pigs, a breed indigenous to China. Through metagenomic analysis, a total of 671 metagenome-assembled genomes (MAGs) were assembled and assigned into 14 bacterial and 1 archaeal phylum, including 97 species from uncultured microbes. The phylum with the highest abundance were identified as Bacillota_A, Bacteroidota, and Bacillota. Remarkably, the investigation revealed nearly 10,000 genes implicated in the degradation of carbohydrates, with a pronounced prevalence within five principal bacterial genera: Prevotella, Cryptobacteroides, Gemmiger, Vescimonas, and Faecousia. Additionally, 87 distinct types of carbohydrate-degrading enzymes were exclusively identified within the gut microbiota of the Lantang pig. These insights not only enhance our understanding of the microbial diversity specific to native Chinese pig breeds but also augment the body of research regarding porcine fiber degradation capabilities. The implications of this study are twofold: it provides strategic directions for optimizing feed efficiency and reducing breeding costs, and it furnishes an expanded gene pool for the microbial synthesis of industrial enzymes in the future. [ABSTRACT FROM AUTHOR]

In our study, we designed and developed a glucose biosensor that operates without a battery or chip. This biosensor utilizes the principles of radio frequency (RF) to operate. For the construction of a glucose-sensitive interdigitated capacitor (IDC), a famous glucose-sensitive substance called phenylboronic acid (PBA) is combined with a polyvinyl chloride (PVC) and n,n-dimethylacetamide (DMAC) solution. According to the theory of radio frequency sensing, the resonance frequency shifts whenever there is a change in the capacitance of the glucose-sensitive IDC. This change is caused by the fluctuations in glucose concentrations. As far as we are aware, this is the first glucose sensor that employs the RF principle to detect changes in glucose solution concentrations using PBA as the principal glucose-sensitive material. The sensor can detect glucose levels with remarkable sensitivity, around 40.89 kHz/decade, and a broad dynamic range covering 10 μM to 1 M. Additionally, the designed biosensor has excellent linearity performance, with a value of around 0.988. The proposed glucose biosensor has several benefits: lightweight, inexpensive, easy to build, and an acceptable selectivity response. Our study concludes by comparing the proposed RF sensor's effectiveness to that of existing glucose sensors, which it outperforms. [ABSTRACT FROM AUTHOR]

Several essential cellular metabolites, such as enzyme cofactors, contain sulfur atoms and their biosynthesis requires specific thiolation enzymes. LarE is an ATP‐dependent sulfur insertase, which catalyzes the sequential conversion of the two carboxylate groups of the precursor of the lactate racemase cofactor into thiocarboxylates. Two types of LarE enzymes are known, one that uses a catalytic cysteine as a sacrificial sulfur donor, and the other one that uses a [4Fe‐4S] cluster as a cofactor. Only the crystal structure of LarE from Lactobacillus plantarum (LpLarE) from the first class has been solved. We report here the crystal structure of LarE from Methanococcus maripaludis (MmLarE), belonging to the second class, in the cluster‐free (apo‐) and cluster‐bound (holo‐) forms. The structure of holo‐MmLarE shows that the [4Fe‐4S] cluster is chelated by three cysteines only, leaving an open coordination site on one Fe atom. Moreover, the fourth nonprotein‐bonded iron atom was able to bind an anionic ligand such as a phosphate group or a chloride ion. Together with the spectroscopic analysis of holo‐MmLarE and the previously reported biochemical investigations of holo‐LarE from Thermotoga maritima, these crystal structures support the hypothesis of a reaction mechanism, in which the [4Fe‐4S] cluster binds a hydrogenosulfide ligand in place of the chloride anion, thus generating a [4Fe‐5S] intermediate, and transfers it to the substrate, as in the case of [4Fe‐4S]‐dependent tRNA thiolation enzymes. [ABSTRACT FROM AUTHOR]

Suicide gene therapy involves introducing viral or bacterial genes into tumor cells, which enables the conversion of a nontoxic prodrug into a toxic‐lethal drug. The application of the bacterial cytosine deaminase (bCD)/5‐fluorocytosine (5‐FC) approach has been beneficial and progressive within the current field of cancer therapy because of the enhanced bystander effect. The basis of this method is the preferential deamination of 5‐FC to 5‐fluorouracil by cancer cells expressing cytosine deaminase (CD), which strongly inhibits DNA synthesis and RNA function, effectively targeting tumor cells. However, the poor binding affinity of toward 5‐FC compared to the natural substrate cytosine and/or inappropriate thermostability limits the clinical applications of this gene therapy approach. Nowadays, many genetic engineering studies have been carried out to solve and improve the activity of this enzyme. In the current review, we intend to discuss the biotechnological aspects of Escherichia coli CD, including its structure, functions, molecular cloning, and protein engineering. We will also explore its relevance in cancer clinical trials. By examining these aspects, we hope to provide a thorough understanding of E. coli CD and its potential applications in cancer therapy. [ABSTRACT FROM AUTHOR]

The field of biosensors is filled with reports and designs of various sensors, with the vast majority focusing on glucose sensing. However, in addition to glucose, there are many other important analytes that are worth investigating as well. In particular, L-amino acids appear as important diagnostic markers for a number of conditions. However, the progress in L-amino acid detection and the development of biosensors for L-amino acids are still somewhat insufficient. In recent years, the need to determine L-amino acids from clinical samples has risen. More clinical data appear to demonstrate that abnormal concentrations of L-amino acids are related to various clinical conditions such as inherited metabolic disorders, dyslipidemia, type 2 diabetes, muscle damage, etc. However, to this day, the diagnostic potential of L-amino acids is not yet fully established. Most likely, this is because of the difficulties in measuring L-amino acids, especially in human blood. In this review article, we extensively investigate the 'overlooked' L-amino acids. We review typical levels of amino acids present in human blood and broadly survey the importance of L-amino acids in most common conditions which can be monitored or diagnosed from changes in L-amino acids present in human blood. We also provide an overview of recent biosensors for L-amino acid monitoring and their advantages and disadvantages, with some other alternative methods for L-amino acid quantification, and finally we outline future perspectives related to the development of biosensing devices for L-amino acid monitoring. [ABSTRACT FROM AUTHOR]

The application of bacteriophages to control foodborne bacterial pathogens in foods has gained traction in recent years. Poultry meat is a major source of Campylobacter jejuni, and a target for the application of bacteriophages. To offer the prospect of a post-harvest control measure, the bacteriophage must function at refrigeration temperatures, where C. jejuni does not grow but can survive. Here, we report actions of three classes of Campylobacter bacteriophage at 4 °C. The pre-incubation of broth cultures at 4 °C before a shift to 42 °C under conditions that support the growth of the host bacteria revealed differences in the time to lysis compared with cultures incubated at 42 °C. The pre-adsorption of the bacteriophage to a sub-population of bacteria is consistent with the observation of asynchronous infection. To ascertain whether the bacteriophages adsorb and infect (the commitment to replicate), we investigated bacteriophage transcription at 4 °C. RNA transcripts for all the bacteriophage host combinations were detected after 15 min, indicating that the interaction is not merely passive. Bacteriophages can infect C. jejuni at refrigeration temperatures, but the infection does not proceed to lysis in the absence of host cell division. [ABSTRACT FROM AUTHOR]

Protein liquid-liquid phase separation (LLPS) is a rapidly emerging field of study on biomolecular condensate formation. In recent years, this phenomenon has been implicated in the process of amyloid fibril formation, serving as an intermediate step between the native protein transition into their aggregated state. The formation of fibrils via LLPS has been demonstrated for a number of proteins related to neurodegenerative disorders, as well as other amyloidoses. Despite the surge in amyloid-related LLPS studies, the influence of protein condensate formation on the end-point fibril characteristics is still far from fully understood. In this work, we compare alpha-synuclein aggregation under different conditions, which promote or negate its LLPS and examine the differences between the formed aggregates. We show that alpha-synuclein phase separation generates a wide variety of assemblies with distinct secondary structures and morphologies. The LLPS-induced structures also possess higher levels of toxicity to cells, indicating that biomolecular condensate formation may be a critical step in the appearance of disease-related fibril variants., (© 2024 Federation of European Biochemical Societies.)

Gene-directed enzyme prodrug therapy is an emerging strategy for cancer treatment based on the delivery of a gene that encodes an enzyme that is able to convert a prodrug into a potent cytotoxin exclusively in target cancer cells. However, it is limited by the lack of suitable enzyme variants and a scarce choice of chemical bonds that could be activated. Therefore, this study is aimed to determine the capability of bacterial amidohydrolases YqfB and D8_RL to activate novel prodrugs and the effect such system has on the viability of eukaryotic cancer cells. We have established cancer cell lines that stably express the bacterial amidohydrolase genes and selected several N4-acylated cytidine derivatives as potential prodrugs. A significant decrease in the viability of HCT116 human colon cancer cell lines expressing either the YqfB or the D8_RL was observed after exposure to the novel prodrugs. The data we acquired suggests that bacterial YqfB and D8_RL amidohydrolases, together with the modified cytidine-based prodrugs, may serve as a promising enzyme-prodrug system for gene-directed enzyme prodrug therapy. [ABSTRACT FROM AUTHOR]

In all domains of life, transfer RNAs (tRNAs) contain post-transcriptionally sulfur-modified nucleosides such as 2- and 4-thiouridine. We have previously reported that a recombinant [4Fe-4S] cluster-containing bacterial desulfidase (TudS) from an uncultured bacterium catalyzes the desulfuration of 2- and 4-thiouracil via a [4Fe-5S] cluster intermediate. However, the in vivo function of TudS enzymes has remained unclear and direct evidence for substrate binding to the [4Fe-4S] cluster during catalysis was lacking. Here, we provide kinetic evidence that 4-thiouridine-5'-monophosphate rather than sulfurated tRNA, thiouracil, thiouridine or 4-thiouridine-5'-triphosphate is the preferred substrate of TudS. The occurrence of sulfur- and substrate-bound catalytic intermediates was uncovered from the observed switch of the S = 3/2 spin state of the catalytic [4Fe-4S] cluster to a S = 1/2 spin state upon substrate addition. We show that a putative gene product from Pseudomonas putida KT2440 acts as a TudS desulfidase in vivo and conclude that TudS-like enzymes are widespread desulfidases involved in recycling and detoxifying tRNA-derived 4-thiouridine monophosphate nucleosides for RNA synthesis. TudS-like enzymes are desulfidases involved in recycling and detoxifying tRNA-derived 4-thiouridine monophosphate nucleosides for RNA synthesis as unveiled by enzymatic, spectroscopic and docking studies of two bacterial TudS enzymes. [ABSTRACT FROM AUTHOR]

This article reviews the recent advances in the field of batteryless near-field communication (NFC) sensors for chemical sensing and biosensing. The commercial availability of low-cost commercial NFC integrated circuits (ICs) and their massive integration in smartphones, used as readers and cloud interfaces, have aroused great interest in new batteryless NFC sensors. The fact that coil antennas are not importantly affected by the body compared with other wireless sensors based on far-field communications makes this technology suitable for future wearable point-of-care testing (PoCT) devices. This review first compares energy harvesting based on NFC to other energy-harvesting technologies. Next, some practical recommendations for designing and tuning NFC-based tags are described. Power transfer is key because in most cases, the energy harvested has to be stable for several seconds and not contaminated by undesired signals. For this reason, the effect of the dimensions of the coils and the conductivity on the wireless power transfer is thoroughly discussed. In the last part of the review, the state of the art in NFC-based chemical and biosensors is presented. NFC-based tags (or sensor tags) are mainly based on commercial or custom NFC ICs, which are used to harvest the energy from the RF field generated by the smartphone to power the electronics. Low-consumption colorimeters and potentiostats can be integrated into these NFC tags, opening the door to the integration of chemical sensors and biosensors, which can be harvested and read from a smartphone. The smartphone is also used to upload the acquired information to the cloud to facilitate the internet of medical things (IoMT) paradigm. Finally, several chipless sensors recently proposed in the literature as a low-cost alternative for chemical applications are discussed. [ABSTRACT FROM AUTHOR]

P-01.1-08 Melanoma therapies compromise signaling processes in the vascular endothelium S. Bromberger 1, M.R. Larsen 2, K. Schossleitner 1 1 Skin & Endothelium Research Division (SERD), Department of Dermatology, Medical Univers... Melanoma and other malignancies often bear mutations in the MAPK pathway - most commonly BRAF SP V600E sp , which contributes to uncontrolled proliferation and survival of tumor cells. The disruption of communication between tumor cells and TAMs could be the base of an efficient anti-cancer strategy to prevent tumor progression. Adamecz 1, P. Bicskei 1, H. Árva 1, N. Igaz 1, É. Veres 2, A. Rónavári 3, A. Gácse... 1 Department of Biochemistry and Molecular Biology, University of Szeged, Szeged, Hungary, 2... Tumor-associated macrophages (TAMs) - as a result of re-programming driven by tumor and other stromal cells - can create and maintain an immunosuppressive environment and promote tumor malignancy. In tumour cells, ER stress is caused by their high degree of translation and metabolism as well as the tumour microenvironment. [Extracted from the article]

One of the biggest challenges faced by food producers is ensuring microbiological safety. Despite strict criteria for food products, foodborne diseases are a global problem and pose a real risk to consumers. Therefore, it is necessary to identify new and more effective methods for eliminating pathogens from food and the food processing environment. According to the European Food Safety Authority (EFSA), the most common foodborne diseases are caused by Campylobacter, Salmonella, Yersinia, Escherichia coli, and Listeria. Out of the five listed, four are Gram‐negative bacteria. Our review focuses on the use of bacteriophages, which are ubiquitous bacterial viruses, and bacteriophage endolysins to eliminate Gram‐negative pathogens. Endolysins cleave specific bonds within the peptidoglycan (PG) of the bacterial cell, causing the cell to burst. Single phages or phage cocktails, which are, in some instances, commercially available products, eliminate pathogenic bacteria in livestock and various food matrices. Endolysins have matured as the most advanced class of antibacterial agents in the clinical sector, but their use in food protection is highly unexplored. Advanced molecular engineering techniques, different formulations, protein encapsulation, and the addition of outer membrane (OM) permeabilization agents enhance the activity of lysins against Gram‐negative pathogens. This creates space for groundbreaking research on the use of lysins in the food sector. [ABSTRACT FROM AUTHOR]

Today, Alzheimer's disease (AD)—the most common neurodegenerative disorder, which affects 50 million people—remains incurable. Several studies suggest that one of the main pathological hallmarks of AD is the accumulation of abnormal amyloid beta (Aβ) aggregates; therefore, many therapeutic approaches focus on anti-Aβ aggregation inhibitors. Taking into consideration that plant-derived secondary metabolites seem to have neuroprotective effects, we attempted to assess the effects of two flavones—eupatorin and scutellarein—on the amyloidogenesis of Aβ peptides. Biophysical experimental methods were employed to inspect the aggregation process of Aβ after its incubation with each natural product, while we monitored their interactions with the oligomerized Aβ through molecular dynamics simulations. More importantly, we validated our in vitro and in silico results in a multicellular organismal model—namely, Caenorhabditis elegans—and we concluded that eupatorin is indeed able to delay the amyloidogenesis of Aβ peptides in a concentration-dependent manner. Finally, we propose that further investigation could lead to the exploitation of eupatorin or its analogues as potential drug candidates. [ABSTRACT FROM AUTHOR]

Interferon induced with helicase C domain-containing protein 1 (IFIH1) gene encodes a cytoplasmic RNA helicase otherwise known as melanoma differentiation-associated 5 (MDA5), a RIG-1-like RNA helicase that recognizes viral RNA and is involved in innate immunity through recognition of viral RNA. Upon binding to double-stranded (ds) RNA, MDA5 forms a filamentous assembly along the length of dsRNA and utilizes molecular signatures to discriminate self, versus non-self on the basis of dsRNA length and methylation. Its missense variant rs35667974 is protective for type 1 diabetes, psoriasis, and psoriatic arthritis, but is also found to be associated with an increased risk for ankylosing spondylitis, Crohn's disease, and ulcerative colitis. To gain insight into the complex role of this variant we performed a structural analysis of MDA5 in complex with dsRNA using molecular dynamics simulations. Our data suggest that while the Ile923Val mutation of the rs35667974 variant does not affect binding to native dsRNA significantly, it displays a destabilizing effect in the presence of 2′-O uridine methylation. Thus, the presence of 2′-O-methylation at the dsRNA introduces a sensing signature that leads to selective reduction of the overall MDA catalytic activity. This study represents an evaluation of the role of the shared rs35667974 variant of autoimmune locus IFIH1, reported to lead to selectively reduced catalytic activity of the modified MDA5 phenotype and, as a consequence, reduced negative feedback on cytokine and chemokine signaling and selectively protection against autoimmunity. [ABSTRACT FROM AUTHOR]

Picolinic acid (PA) is a typical mono-carboxylated pyridine derivative produced by human/animals or microorganisms which could be served as nutrients for bacteria. Most Bordetella strains are pathogens causing pertussis or respiratory disease in humans and/or various animals. Previous studies indicated that Bordetella strains harbor the PA degradation pic gene cluster. However, the degradation of PA by Bordetella strains remains unknown. In this study, a reference strain of genus Bordetella, B. bronchiseptica RB50, was investigated. The organization of pic gene cluster of strain RB50 was found to be similar with that of Alcaligenes faecalis, in which the sequence similarities of each Pic proteins are between 60% to 80% except for PicB2 (47% similarity). The 3,6-dihydroxypicolinic acid (3,6DHPA) decarboxylase gene (BB0271, designated as picCRB50) of strain RB50 was synthesized and over-expressed in E. coli BL21(DE3). The PicCRB50 showed 75% amino acid similarities against known PicC from Alcaligenes faecalis. The purified PicCRB50 can efficiently transform 3,6DHPA to 2,5-dihydroxypyridine. The PicCRB50 exhibits optimal activities at pH 7.0, 35 °C, and the Km and kcat values of PicCRB50 for 3,6DHPA were 20.41 ± 2.60 μM and 7.61 ± 0.53 S−1, respectively. The present study provided new insights into the biodegradation of PA by pathogens of Bordetella spp. [ABSTRACT FROM AUTHOR]

Gene delivery by the pulsed electric field is a promising alternative technology for nonviral transfection; however, the application of short pulses (i.e., nanosecond) is extremely limited. In this work, we aimed to show the capability to improve gene delivery using MHz frequency bursts of nanosecond pulses and characterize the potential use of gold nanoparticles (AuNPs: 9, 13, 14, and 22 nm) in this context. We have used bursts of MHz pulses 3/5/7 kV/cm × 300 ns × 100 and compared the efficacy of the parametric protocols to conventional microsecond protocols (100 µs × 8, 1 Hz) separately and in combination with nanoparticles. Furthermore, the effects of pulses and AuNPs on the generation of reactive oxygen species (ROS) were analyzed. It was shown that gene delivery using microsecond protocols could be significantly improved with AuNPs; however, the efficacy is strongly dependent on the surface charge of AuNPs and their size. The capability of local field amplification using AuNPs was also confirmed by finite element method simulation. Finally, it was shown that AuNPs are not effective with nanosecond protocols. However, MHz protocols are still competitive in the context of gene delivery, resulting in low ROS generation, preserved viability, and easier procedure to trigger comparable efficacy. [ABSTRACT FROM AUTHOR]

The 5% addition of biochar in composting in rows contributes significantly to reducing volatile organic compound(VOC) emissions. When composting with the addition of biochar, the average temperature increased by 13 ± 6.7 °C during the whole period, and the thermophilic phase was extended by 11 days. The higher temperature supported a reduction in the time necessary for achieving the biological stability observed by the oxygen uptake rate by more than 10 days. For organic compounds formed by the degradation of easily degradable primary components (proteins), the addition of biochar significantly reduces the release of organic compounds with heterocyclically bound nitrogen (Norg-VOCs) and volatile sulfur compounds (VSCs). The end of the biodegradation process is indicated by a decrease in VOC concentrations below initial values in the input material. This state was achieved in the compost with added biochar after 47 days, while in compost without added biochar, it lasted 60 days. [ABSTRACT FROM AUTHOR]

A plethora of di- and oligosaccharides isolated from the natural sources are used in food and pharmaceutical industry. An enzymatic hydrolysis of fungal cell wall β-glucans is a good alternative to produce the desired oligosaccharides with different functionalities, such as the flavour enhancer gentiobiose. We have previously identified PsGly30A as a potential yeast cell wall degrading β-1,6-glycosidase. The aim of this study is to characterise the PsGly30A enzyme, a member of the GH30 family, and to evaluate its suitability for the production of gentiobiose from β-1,6-glucans. An endo-β-1,6-glucanase PsGly30A encoding gene from Paenibacillus sp. GKG has been cloned and overexpressed in Escherichia coli. The recombinant enzyme has been active towards pustulan and yeast β-glucan, but not on laminarin from the Laminaria digitata, confirming the endo-β-1,6-glucanase mode of action. The PsGly30A shows the highest activity at pH 5.5 and 50 °C. The specific activity of PsGly30A on pustulan (1262±82 U/mg) is among the highest reported for GH30 β-1,6-glycosidases. Moreover, gentiobiose is the major reaction product when pustulan, yeast β-glucan or yeast cell walls have been used as a substrate. Therefore, PsGly30A is a promising catalyst for valorisation of the yeast-related by-products., (© 2024 Wiley‐VCH GmbH.)

Brevibacillus laterosporus (Bl) is a Gram-positive and spore-forming bacterium belonging to the Brevibacillus brevis phylogenetic cluster. Globally, insect pathogenic strains of the bacterium have been isolated, characterised, and some activities have been patented. Two isolates, Bl 1821L and Bl 1951, exhibiting pathogenicity against the diamondback moth and mosquitoes, are under development as a biopesticide in New Zealand. However, due to the suspected activity of putative antibacterial proteins (ABPs), the endemic isolates often grow erratically. Various purification methods, including size exclusion chromatography, sucrose density gradient centrifugation, polyethylene glycol precipitation, and ammonium sulphate precipitation employed in this study, enabled the isolation of two putative antibacterial proteins of ~30 and ~48 kD from Bl 1821L and one putative antibacterial protein of ~30 kD from Bl 1951. Purification of the uninduced cultures of Bl 1821L and Bl 1951 also yielded protein bands of ~30 and ~48 kD on SDS-PAGE, which indicated their spontaneous induction. A disc diffusion assay was used to determine the antagonistic activities of the putative ABPs. Subsequent transmission electron microscope (TEM) examination of a purified putative antibacterial protein-containing solution showed the presence of encapsulin (~30 kD) and polysheath (~48 kD)-like structures. Although only the ~30 kD protein was purified from Bl 1951, both structures were seen in this strain under TEM. Furthermore, while assessing the antibacterial activity of some fractions of Bl 1951 against Bl 1821L in the size exclusion chromatography method, the population of Bl 1821L persister cells was noted. Overall, this work added a wealth of knowledge about the purification of the high-molecular-weight (HMW) proteins (bacteriocins) of Gram-positive bacteria including Bl. [ABSTRACT FROM AUTHOR]

Amyloidogenic protein/peptide aggregation into fibrillar aggregates is associated with multiple amyloidoses, including widespread neurodegenerative disorders. Despite years of research and a well-understood mechanism, there are still very few treatments available for the increasing number of amyloid-related disorders. In recent years, the search for potential anti-aggregation compounds has shifted toward naturally occurring molecules, with one of the most promising being epigallocatechin-3-gallate (EGCG). This polyphenolic compound was shown to inhibit the aggregation of several amyloidogenic proteins/peptides, including amyloid-beta (related to Alzheimer's disease) and alpha-synuclein (related to Parkinson's disease). However, multiple reports have indicated its limited stability under physiological conditions and the possibility of EGCG autoxidation products being the actual inhibitory compounds. In this work, we explore how different EGCG autoxidation products associate with non-aggregated insulin, as well as how they affect its aggregation and resulting fibril structure. We also show that there is a specific incubation time required for the emergence of compounds, which alters the amyloid aggregation process. [ABSTRACT FROM AUTHOR]

The widespread superfamily of the human activating signal cointegrator homology (ASCH) domain was identified almost 20 years ago; however, the amount of experimental data regarding the biological function of the domain is scarce. With this study, we aimed to determine the putative cellular functions of four hypothetical ASCH domain-containing amidohydrolase YqfB analogues by investigating their activity towards various N-acylated cytosine derivatives, including potential nucleoside-derived prodrugs, as well as their ability to bind/degrade nucleic acids in vitro. According to determined kinetic parameters, N4-acetylcytidine is assumed to be the primary substrate for amidohydrolases. Despite the similarity to the proteins containing the PUA domain, no nucleic acid binding activity was detected for YqfB-like proteins, suggesting that, in vivo, these enzymes are a part of the pyrimidine salvage pathway. We also demonstrate the possibility of the expression of YqfB-type amidohydrolases in both prokaryotic and eukaryotic hosts. The small protein size and remarkable halotolerance of YqfB-type amidohydrolases are of great interest for further fundamental research and biotechnological applications [ABSTRACT FROM AUTHOR]

Sulfur is an essential element for a variety of cellular constituents in all living organisms and adds considerable functionality to a wide range of biomolecules. The pathways for incorporating sulfur into central metabolites of the cell such as cysteine, methionine, cystathionine, and homocysteine have long been established. Furthermore, the importance of persulfide intermediates during the biosynthesis of thionucleotide-containing tRNAs, iron-sulfur clusters, thiamin diphosphate, and the molybdenum cofactor are well known. This review briefly surveys these topics while emphasizing more recent aspects of sulfur metabolism that involve unconventional biosynthetic pathways. Sacrificial sulfur transfers from protein cysteinyl side chains to precursors of thiamin and the nickel-pincer nucleotide (NPN) cofactor are described. Newer aspects of synthesis for lipoic acid, biotin, and other compounds are summarized, focusing on the requisite iron-sulfur cluster destruction. Sulfur transfers by using a noncore sulfide ligand bound to a [4Fe-4S] cluster are highlighted for generating certain thioamides and for alternative biosynthetic pathways of thionucleotides and the NPN cofactor. Thioamide formation by activating an amide oxygen atom via phosphorylation also is illustrated. The discussion of these topics stresses the chemical reaction mechanisms of the transformations and generally avoids comments on the gene/protein nomenclature or the sources of the enzymes. This work sets the stage for future efforts to decipher the diverse mechanisms of sulfur incorporation into biological molecules. [ABSTRACT FROM AUTHOR]

Protein aggregation in the form of amyloid fibrils is linked with the onset and progression of more than 30 amyloidoses, including multiple neurodegenerative disorders, such as Alzheimer's or Parkinson's disease. Despite countless studies and years of research, the process of such aggregate formation is still not fully understood. One peculiar aspect of amyloids is that they appear to be capable of undergoing structural rearrangements even after the fibrils have already formed. Such a phenomenon was reported to occur in the case of alpha-synuclein and amyloid beta aggregates after a long period of incubation. In this work, we examine whether incubation at an elevated temperature can induce the restructurization of four different conformation alpha-synuclein amyloid fibrils. We show that this structural alteration occurs in a relatively brief time period, when the aggregates are incubated at 60 °C. Additionally, it appears that during this process multiple conformationally-distinct alpha-synuclein fibrils all shift towards an identical secondary structure. [ABSTRACT FROM AUTHOR]

Burkholderia ambifaria T16 is a bacterium isolated from the rhizosphere of barley plants that showed a remarkable antifungal activity. This strain was also able to degrade fusaric acid (5-Butylpyridine-2-carboxylic acid) and detoxify this mycotoxin in inoculated barley seedlings. Genes and enzymes responsible for fusaric acid degradation have an important biotechnological potential in the control of fungal diseases caused by fusaric acid producers, or in the biodegradation/bio catalysis processes of pyridine derivatives. In this study, the complete genome of B. ambifaria T16 was sequenced and analyzed to identify genes involved in survival and competition in the rhizosphere, plant growth promotion, fungal growth inhibition, and degradation of aromatic compounds. The genomic analysis revealed the presence of several operons for the biosynthesis of antimicrobial compounds, such as pyrrolnitrin, ornibactin, occidiofungin and the membrane-associated AFC-BC11. These compounds were also detected in bacterial culture supernatants by mass spectrometry analysis. In addition, this strain has multiple genes contributing to its plant growth-promoting profile, including those for acetoin, 2,3-butanediol and indole-3-acetic acid production, siderophores biosynthesis, and solubilisation of organic and inorganic phosphate. A pan-genomic analysis demonstrated that the genome of strain T16 possesses large gene clusters that are absent in the genomes of B. ambifaria reference strains. According to predictions, most of these clusters would be involved in aromatic compounds degradation. One genomic region, encoding flavin-dependent monooxygenases of unknown function, is proposed as a candidate responsible for fusaric acid degradation. [ABSTRACT FROM AUTHOR]