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For the first time, a Gram-positive microorganism capable of degrading 4-hydroxypyridine was isolated by enrichment techniques from soil. The isolate IN13 utilizes also 2-hydroxypyridine as a sole carbon, nitrogen and energy source. During the degradation of 2-hydroxypyridine a soluble blue pigment was accumulated in the culture. The 16S rRNA-based phylogeny analysis has indicated that the IN13 isolate belongs to the genus Arthrobacter and with Arthrobacter sp. A4Z24, Arthrobacter sp. A1-1, and several uncultured strains forms a separate group. The biodegradation of 4-hydroxypyridine and 2-hydroxypyridine was investigated. Biodegradation studies with washed cells confirmed 4-hydroxypyridine and 2-hydroxypyridine consumption. IN13 growing in a medium with 2-hydroxypyridine tends to lose its 4-hydroxypyridine degrading activity at a significant rate. [ABSTRACT FROM AUTHOR]

Enzymatic degradation of plastic pollution offers a promising environmentally friendly waste management strategy, however, suitable biocatalysts must be screened and developed. Traditional screening methods using soluble or solubilised polymers do not necessarily identify enzymes that are effective against solid or crystalline polymers. This study presents a simple, time-saving and cost-effective method for identifying microorganisms and enzymes capable of degrading polymeric films. The method was tested on polycaprolactone (PCL), polyethylene terephthalate (PET), polylactate (PLA) and polyhydroxybutyrate/polyhydroxyvalerate (PHB/PHV) films. It involves two steps: first, screening for PCL diol (PCLD)-degrading microorganisms on agar plates, and second, testing these microorganisms on polyester films. Using this screening method, over 100 PCLD-degrading microorganisms and 27 E. coli clones carrying genomic or metagenomic DNA fragments have been isolated. In addition, recombinant cutinases from Streptomyces scabiei and Thermobifida fusca have been tested. Approximately 66 % of the microorganisms forming halos on PCLD agar plates hydrolysed PCL and 6 % - the biaxially oriented PET film. In addition, five PLA- and four PHB/PHV-degrading esterases have been identified. The proposed method is effective for detecting both wild-type and recombinant microorganisms, as well as recombinant enzymes from in vitro transcription-translation reactions. Screening for thermostable and thermophilic enzymes, including those resistant to organic solvents or environmental inhibitors, is also easily implemented., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Nina Urbeliene, Renata Gasparaviciute, Renata Gudiukaite and Rolandas Meskys has patent #EP23187384.5 pending to Vilnius University. If there are other authors, they 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 © 2025 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

Cytidine deaminases (CDAs) catalyze the hydrolytic deamination of cytidine and 2'-deoxycytidine to uridine and 2'-deoxyuridine. Here, we report that prokaryotic homo-tetrameric CDAs catalyze the nucleophilic substitution at the fourth position of N 4 -acyl-cytidines, N 4 -alkyl-cytidines, and N 4 -alkyloxycarbonyl-cytidines, and S 4 -alkylthio-uridines and O 4 -alkyl-uridines, converting them to uridine and corresponding amide, amine, carbamate, thiol, or alcohol as leaving groups. The x-ray structure of a metagenomic CDA_F14 and the molecular modeling of the CDAs used in this study show a relationship between the bulkiness of a leaving group and the volume of the binding pocket, which is partly determined by the flexible β3α3 loop of CDAs. We propose that CDAs that are active toward a wide range of substrates participate in salvage and/or catabolism of variously modified pyrimidine nucleosides. This identified promiscuity of CDAs expands the knowledge about the cellular turnover of cytidine derivatives, including the pharmacokinetics of pyrimidine-based prodrugs.

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.

Here, we present an improved whole-cell biocatalysis system for the synthesis of heteroaromatic N-oxides based on the production of a soluble di-iron monooxygenase PmlABCDEF in Pseudomonas sp. MIL9 and Pseudomonas putida KT2440. The presented biocatalysis system performs under environmentally benign conditions, features a straightforward and inexpensive procedure and possesses a high substrate conversion and product yield. The capacity of gram-scale production was reached in the simple shake-flask cultivation. The template substrates (pyridine, pyrazine, 2-aminopyrimidine) have been converted into pyridine-1-oxide, pyrazine-1-oxide and 2-aminopyrimidine-1-oxide in product titres of 18.0, 19.1 and 18.3 g l -1 , respectively. To our knowledge, this is the highest reported productivity of aromatic N-oxides using biocatalysis methods. Moreover, comparing to the chemical method of aromatic N-oxides synthesis based on meta-chloroperoxybenzoic acid, the developed approach is applicable for a regioselective oxidation that is an additional advantageous option in the preparation of the anticipated N-oxides., (© 2021 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

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.)

N-Heterocyclic compounds are widely spread in the biosphere, being constituents of alkaloids, cofactors, allelochemicals, and artificial substances. However, the fate of such compounds including a catabolism of hydroxylated pyridines is not yet fully understood. Arthrobacter sp. IN13 is capable of using 4-hydroxypyridine as a sole source of carbon and energy. Three substrate-inducible proteins were detected by comparing protein expression profiles, and peptide mass fingerprinting was performed using MS/MS. After partial sequencing of the genome, we were able to locate genes encoding 4-hydroxypyridine-inducible proteins and identify the kpi gene cluster consisting of 16 open reading frames. The recombinant expression of genes from this locus in Escherichia coli and Rhodococcus erytropolis SQ1 allowed an elucidation of the biochemical functions of the proteins. We report that in Arthrobacter sp. IN13, the initial hydroxylation of 4-hydroxypyridine is catalyzed by a flavin-dependent monooxygenase (KpiA). A product of the monooxygenase reaction is identified as 3,4-dihydroxypyridine, and a subsequent oxidative opening of the ring is performed by a hypothetical amidohydrolase (KpiC). The 3-( N -formyl)-formiminopyruvate formed in this reaction is further converted by KpiB hydrolase to 3-formylpyruvate. Thus, the degradation of 4-hydroxypyridine in Arthrobacter sp. IN13 was analyzed at genetic and biochemical levels, elucidating this catabolic pathway., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Here, we present a proof-of-principle for a new high-throughput functional screening of metagenomic libraries for the selection of enzymes with different activities, predetermined by the substrate being used. By this approach, a total of 21 enzyme-coding genes were selected, including members of xanthine dehydrogenase, aldehyde dehydrogenase (ALDH), and amidohydrolase families. The screening system is based on a pro-chromogenic substrate, which is transformed by the target enzyme to indole-3-carboxylic acid. The later compound is converted to indoxyl by a newly identified indole-3-carboxylate monooxygenase (Icm). Due to the spontaneous oxidation of indoxyl to indigo, the target enzyme-producing colonies turn blue. Two types of pro-chromogenic substrates have been tested. Indole-3-carboxaldehydes and the amides of indole-3-carboxylic acid have been applied as substrates for screening of the ALDHs and amidohydrolases, respectively. Both plate assays described here are rapid, convenient, easy to perform, and adaptable for the screening of a large number of samples both in Escherichia coli and Rhodococcus sp. In addition, the fine-tuning of the pro-chromogenic substrate allows screening enzymes with the desired substrate specificity., (© 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

A high-throughput method (≥ 10 6 of clones can be analysed on a single agar plate) for the selection of ester-hydrolysing enzymes was developed based on the uridine auxotrophy of Escherichia coli strain DH10B ΔpyrFEC and the acylated derivatives 2',3',5'-O-tri-acetyluridine and 2',3',5'-O-tri-hexanoyluridine as the sole source of uridine. The proposed approach permits the selection of hydrolases belonging to different families and active towards different substrates. Moreover, the ester group of the substrate used for the selection, at least partly, determined the specificity of the selected enzymes., (© 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Microbial degradation of 2-hydroxypyridine usually results in the formation of a blue pigment (nicotine blue). In contrast, the Burkholderia sp. strain MAK1 bacterium utilizes 2-hydroxypyridine without the accumulation of nicotine blue. This scarcely investigated degradation pathway presumably employs 2-hydroxypyridine 5-monooxygenase, an elusive enzyme that has been hypothesized but has yet to be identified or characterized. The isolation of the mutant strain Burkholderia sp. MAK1 ΔP5 that is unable to utilize 2-hydroxypyridine has led to the identification of a gene cluster (designated hpd ) which is responsible for the degradation of 2-hydroxypyridine. The activity of 2-hydroxypyridine 5-monooxygenase has been assigned to a soluble diiron monooxygenase (SDIMO) encoded by a five-gene cluster ( hpdA , hpdB , hpdC , hpdD , and hpdE ). A 4.5-kb DNA fragment containing all five genes has been successfully expressed in Burkholderia sp. MAK1 ΔP5 cells. We have proved that the recombinant HpdABCDE protein catalyzes the enzymatic turnover of 2-hydroxypyridine to 2,5-dihydroxypyridine. Moreover, we have confirmed that emerging 2,5-dihydroxypyridine is a substrate for HpdF, an enzyme similar to 2,5-dihydroxypyridine 5,6-dioxygenases that are involved in the catabolic pathways of nicotine and nicotinic acid. The proteins and genes identified in this study have allowed the identification of a novel degradation pathway of 2-hydroxypyridine. Our results provide a better understanding of the biodegradation of pyridine derivatives in nature. Also, the discovered 2-hydroxypyridine 5-monooxygenase may be an attractive catalyst for the regioselective synthesis of various N -heterocyclic compounds. IMPORTANCE The degradation pathway of 2-hydroxypyridine without the accumulation of a blue pigment is relatively unexplored, as, to our knowledge, no genetic data related to this process have ever been presented. In this paper, we describe genes and enzymes involved in this little-studied catabolic pathway. This work provides new insights into the metabolism of 2-hydroxypyridine in nature. A broad-range substrate specificity of 2-hydroxypyridine 5-monooxygenase, a key enzyme in the degradation, makes this biocatalyst attractive for the regioselective hydroxylation of pyridine derivatives., (Copyright © 2018 American Society for Microbiology.)

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.)

Indole is a molecule of considerable biochemical significance, acting as both an interspecies signal molecule and a building block of biological elements. Bacterial indole degradation has been demonstrated for a number of cases; however, very little is known about genes and proteins involved in this process. This study reports the cloning and initial functional characterization of genes ( iif and ant cluster) responsible for indole biodegradation in Acinetobacter sp. strain O153. The catabolic cascade was reconstituted in vitro with recombinant proteins, and each protein was assigned an enzymatic function. Degradation starts with oxidation, mediated by the IifC and IifD flavin-dependent two-component oxygenase system. Formation of indigo is prevented by IifB, and the final product, anthranilic acid, is formed by IifA, an enzyme which is both structurally and functionally comparable to cofactor-independent oxygenases. Moreover, the iif cluster was identified in the genomes of a wide range of bacteria, suggesting the potential of widespread Iif-mediated indole degradation. This work provides novel insights into the genetic background of microbial indole biodegradation. IMPORTANCE The key finding of this research is identification of the genes responsible for microbial biodegradation of indole, a toxic N -heterocyclic compound. A large amount of indole is present in urban wastewater and sewage sludge, creating a demand for an efficient and eco-friendly means to eliminate this pollutant. A common strategy of oxidizing indole to indigo has the major drawback of producing insoluble material. Genes and proteins of Acinetobacter sp. strain O153 (DSM 103907) reported here pave the way for effective and indigo-free indole removal. In addition, this work suggests possible novel means of indole-mediated bacterial interactions and provides the basis for future research on indole metabolism., (Copyright © 2017 American Society for Microbiology.)

Pyridinols and pyridinamines are important intermediates with many applications in chemical industry. The pyridine derivatives are in great demand as synthons for pharmaceutical products. Moreover, pyridines are used either as biologically active substances or as building blocks for polymers with unique physical properties. Application of enzymes or whole cells is an attractive strategy for preparation of hydroxylated pyridines since the methods for chemical synthesis of pyridinols, particularly aminopyridinols, are usually limited or inefficient. Burkholderia sp. MAK1 (DSM102049), capable of using pyridin-2-ol as the sole carbon and energy source, was isolated from soil. Whole cells of Burkholderia sp. MAK1 were confirmed to possess a good ability to convert different pyridin-2-amines and pyridin-2-ones into their 5-hydroxy derivatives. Moreover, several methylpyridines as well as methylated pyrazines were converted to appropriate N-oxides. In conclusion, regioselective oxyfunctionalization of pyridine derivatives using whole cells of Burkholderia sp. MAK1 is a promising method for the preparation of various pyridin-5-ols and pyridin-N-oxides.

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.)

A bacterial strain 5HP capable of degrading and utilizing 5-hydroxypicolinic acid as the sole source of carbon and energy was isolated from soil. In addition, the isolate 5HP could also utilize 3-hydroxypyridine and 3-cyanopyridine as well as nicotinic, benzoic and p-hydroxybenzoic acids for growth in the basic salt media. On the basis of 16S rRNA gene sequence analysis, the isolate 5HP was shown to belong to the genus Pusillimonas. Both the bioconversion analysis using resting cells and the enzymatic assay showed that the degradation of 5-hydroxypicolinic acid, 3-hydroxypyridine and nicotinic acid was inducible and proceeded via formation of the same metabolite, 2,5-dihydroxypyridine. The activity of a novel enzyme, 5-hydroxypicolinate 2-monooxygenase, was detected in the cell-free extracts prepared from 5-hydroxypicolinate-grown cells. The enzyme was partially purified and was shown to catalyze the oxidative decarboxylation of 5-hydroxypicolinate to 2,5-dihydroxypyridine. The activity of 5-hydroxypicolinate 2-monooxygenase was dependent on O2, NADH and FAD.