Soluble Di-iron Monooxygenase Research Articles

Unravelling the role of the group 6 soluble di-iron monooxygenase (SDIMO) SmoABCD in alkane metabolism and chlorinated alkane degradation.

Soluble di-iron monooxygenases (SDIMOs) are multi-component enzymes catalysing the oxidation of various substrates. These enzymes are characterized by high sequence and functional diversity that is still not well understood despite their key role in biotechnological processes including contaminant biodegradation. In this study, we analysed a mutant of Rhodoccocus aetherivorans BCP1 (BCP1-2.10) characterized by a transposon insertion in the gene smoA encoding the alpha subunit of the plasmid-located SDIMO SmoABCD. The mutant BCP1-2.10 showed a reduced capacity to grow on propane, lost the ability to grow on butane, pentane and n-hexane and was heavily impaired in the capacity to degrade chloroform and trichloroethane. The expression of the additional SDIMO prmABCD in BCP1-2.10 probably allowed the mutant to partially grow on propane and to degrade it, to some extent, together with the other short-chain n-alkanes. The complementation of the mutant, conducted by introducing smoABCD in the genome as a single copy under a constitutive promoter or within a plasmid under a thiostreptone-inducible promoter, allowed the recovery of the alkanotrophic phenotype as well as the capacity to degrade chlorinated n-alkanes. The heterologous expression of smoABCD allowed a non-alkanotrophic Rhodococcus strain to grow on pentane and n-hexane when the gene cluster was introduced together with the downstream genes encoding alcohol and aldehyde dehydrogenases and a GroEL chaperon. BCP1 smoA gene was shown to belong to the group 6 SDIMOs, which is a rare group of monooxygenases mostly present in Mycobacterium genus and in a few Rhodococcus strains. SmoABCD originally evolved in Mycobacterium and was then acquired by Rhodococcus through horizontal gene transfer events. This work extends the knowledge of the biotechnologically relevant SDIMOs by providing functional and evolutionary insights into a group 6 SDIMO in Rhodococcus and demonstrating its key role in the metabolism of short-chain alkanes and degradation of chlorinated n-alkanes.

A novel soluble di-iron monooxygenase from the soil bacterium Solimonas soli.

Soluble di-iron monooxygenase (SDIMO) enzymes enable insertion of oxygen into diverse substrates and play significant roles in biogeochemistry, bioremediation and biocatalysis. An unusual SDIMO was detected in an earlier study in the genome of the soil organism Solimonas soli, but was not characterized. Here, we show that the S. soli SDIMO is part of a new clade, which we define as 'Group 7'; these share a conserved gene organization with alkene monooxygenases but have only low amino acid identity. The S. soli genes (named zmoABCD) could be functionally expressed in Pseudomonas putida KT2440 but not in Escherichia coli TOP10. The recombinants made epoxides from C2 C8 alkenes, preferring small linear alkenes (e.g. propene), but also epoxidating branched, carboxylated and chlorinated substrates. Enzymatic epoxidation of acrylic acid was observed for the first time. ZmoABCD oxidised the organochlorine pollutants vinyl chloride (VC) and cis-1,2-dichloroethene (cDCE), with the release of inorganic chloride from VC but not cDCE. The original host bacterium S. soli could not grow on any alkenes tested but grew well on phenol and n-octane. Further work is needed to link ZmoABCD and the other Group 7 SDIMOs to specific physiological and ecological roles.

A multicomponent propane monooxygenase catalyzes the initial degradation of methyl tert-butyl ether in Mycobacterium vaccae JOB5.

Methyl tert-butyl ether (MTBE) has been recognized as a groundwater contaminant due to its widespread distribution and potential threat to human health. The limited understanding of the enzymes catalyzing MTBE degradation restricts their application in MTBE bioremediation. In this study, an MTBE-degrading soluble di-iron monooxygenase that clusters phylogenetically with a known propane monooxygenase (PRM) encoded by the prmABCD gene cluster was identified and functionally characterized, revealing their role in MTBE metabolism by Mycobacterium vaccae JOB5. Transcriptome analysis demonstrated that the expression of prmABCD was upregulated when JOB5 was induced by MTBE. Escherichia coli Rosetta heterologously expressing prmABCD from JOB5 could transform MTBE, indicating that the PRM of JOB5 is capable of the initial degradation of MTBE. The loss of the gene encoding the oxygenase α-subunit or β-subunit, the coupling protein, or the reductase disrupted MTBE transformation by the recombinant E. coli Rosetta. In addition, the catalytic capacity of PRM is likely affected by residue G95 in the active site pocket and residues I84, P165, A269, and V270 in the substrate tunnel structure. Mutation of amino acids in the active site and substrate tunnel resulted in inefficiency or inactivation of MTBE degradation, and the activity in 1,4-dioxane (1,4-D) degradation was diminished less than that in MTBE degradation.IMPORTANCEMulticomponent monooxygenases catalyzing the initial hydroxylation of MTBE are important in MTBE biodegradation. Previous studies of MTBE degradation enzymes have focused on P450s, alkane monooxygenase and MTBE monooxygenase, but the vital role of soluble di-iron monooxygenases has rarely been reported. In this study, we deciphered the essential catalytic role of a PRM and revealed the key residues of the PRM in MTBE metabolism. Our findings provide new insight into the MTBE-degrading gene cluster and enzymes in bacteria. This characterization of the PRM associated with MTBE degradation expands our understanding of MTBE-degrading gene diversity and provides a novel candidate enzyme for the bioremediation of MTBE-contaminated sites.

The 2-methylpropene degradation pathway in Mycobacteriaceae family strains.

Mycolicibacterium gadium IBE100 and Mycobacterium paragordonae IBE200 are aerobic, chemoorganoheterotrophic bacteria isolated from activated sludge from a wastewater treatment plant. They use 2-methylpropene (isobutene, 2-MP) as the sole source of carbon and energy. Here, we postulate a degradation pathway of 2-methylpropene derived from whole genome sequencing, differential expression analysis and peptide-mass fingerprinting. Key genes identified are coding for a 4-component soluble diiron monooxygenase with epoxidase activity, an epoxide hydrolase, and a 2-hydroxyisobutyryl-CoA mutase. In both strains, involved genes are arranged in clusters of 61.0 and 58.5 kbp, respectively, which also contain the genes coding for parts of the aerobic pathway of adenosylcobalamin synthesis. This vitamin is essential for the carbon rearrangement reaction catalysed by the mutase. These findings provide data for the identification of potential 2-methylpropene degraders.

Effectiveness of tetrahydrofuran at enhancing the 1,4-dioxane degradation ability of activated sludge lacking prior exposure to 1,4-dioxane.

1,4-dioxane (DX) is a contaminant of emerging concern in water environments. The enrichment of DX-degrading bacteria indigenous to activated sludge is key for the efficient biological removal of DX in wastewater. To identify an effective substrate, which enables the selective enrichment of DX-degrading bacteria and has lower toxicity and persistence than DX, this study explored the effectiveness of tetrahydrofuran (THF) at enhancing the DX degradation ability of activated sludge without historical exposure to DX. Although the activated sludge initially exhibited negligible ability to degrade DX (100 mg-C/L) as the sole carbon source, the repeated batch cultivation on THF could enrich bacterial populations capable of degrading DX, inducing the DX degradation ability in activated sludge as effectively as DX did. The THF-enrichment culture after 4 weeks degraded 100 mg-C/L DX almost completely within 21 d. Sequencing analyses revealed that soluble di-iron monooxygenase group 5C, including THF/DX monooxygenase, would play a dominant role in the initial oxidation of DX in THF-enrichment culture, which completely differed from the enrichment culture cultivated on DX. The results indicate that THF can be applied as an effective substrate to enhance the DX degradation ability of microbial consortia, irrespective of the intrinsic ability.

Identification of active gaseous-alkane degraders at natural gas seeps

Natural gas seeps release significant amounts of methane and other gases including ethane and propane contributing to global climate change. In this study, bacterial actively consuming short-chain alkanes were identified by cultivation, whole-genome sequencing, and stable-isotope probing (SIP)-metagenomics using 13C-propane and 13C-ethane from two different natural gas seeps, Pipe Creek and Andreiasu Everlasting Fire. Nearly 100 metagenome-assembled genomes (MAGs) (completeness 70–99%) were recovered from both sites. Among these, 16 MAGs had genes encoding the soluble di-iron monooxygenase (SDIMO). The MAGs were affiliated to Actinobacteria (two MAGs), Alphaproteobacteria (ten MAGs), and Gammaproteobacteria (four MAGs). Additionally, three gaseous-alkane degraders were isolated in pure culture, all of which could grow on ethane, propane, and butane and possessed SDIMO-related genes. Two Rhodoblastus strains (PC2 and PC3) were from Pipe Creek and a Mycolicibacterium strain (ANDR5) from Andreiasu. Strains PC2 and PC3 encoded putative butane monooxygenases (MOs) and strain ANDR5 contained a propane MO. Mycolicibacterium strain ANDR5 and MAG19a, highly abundant in incubations with 13C-ethane, share an amino acid identity (AAI) of 99.3%. We show using a combination of enrichment and isolation, and cultivation-independent techniques, that these natural gas seeps contain a diverse community of active bacteria oxidising gaseous-alkanes, which play an important role in biogeochemical cycling of natural gas.

An efficient and regioselective biocatalytic synthesis of aromatic N-oxides by using a soluble di-iron monooxygenase PmlABCDEF produced in the Pseudomonas species.

SummaryHere, 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.

Draft Genome Sequences of Four Aerobic Isobutane-Metabolizing Bacteria.

Here, we report the draft genome sequences of four aerobic gaseous alkane-oxidizing bacteria isolated from soil by enrichment culture using isobutane (2-methylpropane) as the sole carbon and energy source. The sequences all reveal microorganisms with multiple alkane-oxidizing monooxygenases, including soluble di-iron monooxygenases (SDIMOs), copper-containing monooxygenases (CuMMOs), and alkane hydroxylases (AHs).

Isolation and Characterization of Novel Bacteria Capable of Degrading 1,4-Dioxane in the Presence of Diverse Co-Occurring Compounds.

Biodegradation is found to be a promising, cost-effective and eco-friendly option for the treatment of industrial wastewater contaminated by 1,4-dioxane (1,4-D), a highly stable synthetic chemical and probable human carcinogen. This study aimed to isolate, identify, and characterize metabolic 1,4-D-degrading bacteria from a stable 1,4-D-degrading microbial consortium. Three bacterial strains (designated as strains TS28, TS32, and TS43) capable of degrading 1,4-D as a sole carbon and energy source were isolated and identified as Gram-positive Pseudonocardia sp. (TS28) and Gram-negative Dokdonella sp. (TS32) and Afipia sp. (TS43). This study, for the first time, confirmed that the genus Dokdonella is involved in the biodegradation of 1,4-D. The results reveal that all of the isolated strains possess inducible 1,4-D-degrading enzymes and also confirm the presence of a gene encoding tetrahydrofuran/dioxane monooxygenase (thmA/dxmA) belonging to group 5 soluble di-iron monooxygenases (SDIMOs) in both genomic and plasmid DNA of each of the strains, which is possibly responsible for the initial oxidation of 1,4-D. Moreover, the isolated strains showed a broad substrate range and are capable of degrading 1,4-D in the presence of additional substrates, including easy-to-degrade compounds, 1,4-D biodegradation intermediates, structural analogs, and co-contaminants of 1,4-D. This indicates the potential of the isolated strains, especially strain TS32, in removing 1,4-D from contaminated industrial wastewater containing additional organic load. Additionally, the results will help to improve our understanding of how multiple 1,4-D-degraders stably co-exist and interact in the consortium, relying on a single carbon source (1,4-D) in order to develop an efficient biological 1,4-D treatment system.

Degradation of 1,4-Dioxane by Xanthobacter sp. YN2.

1,4-Dioxane is a highly toxic and carcinogenic pollutant found worldwide in groundwater and soil environments. Several microorganisms have been isolated by their ability to grow on 1,4-dioxane; however, low 1,4-dioxane tolerance and slow degradation kinetics remain obstacles for their use in 1,4-dioxane bioremediation. We report here the isolation and characterization of a new strain, Xanthobacter sp. YN2, capable of highly efficient 1,4-dioxane degradation. High degradation efficiency and high tolerance to 1,4-dioxane make this new strain an ideal candidate for the biodegradation of 1,4-dioxane in various treatment facilities. The maximum degradation rate of 1,4-dioxane was found to be 1.10 mg-1,4-dioxane/h mg-protein. Furthermore, Xanthobacter sp. YN2 was shown to grow in the presence of higher than 3000mg/L 1,4-dioxane with little to no degradation inhibition. In addition, Xanthobacter sp. YN2 could grow on and degrade 1,4-dioxane at pH ranges 5 to 8 and temperatures between 20 and 40°C. Xanthobacter sp. YN2 was also found to be able to grow on a variety of other substrates including several analogs of 1,4-dioxane. Genome sequence analyses revealed the presence of two soluble di-iron monooxygenase (SDIMO) gene clusters, and regulation studies determined that all of the genes in these two clusters were upregulated in the presence of 1,4-dioxane. This study provides insights into the bacterial stress response and the highly efficient biodegradation of 1,4-dioxane as well as the identification of a novel Group-2 SDIMO.

Isoprene Oxidation by the Gram-Negative Model bacterium Variovorax sp. WS11.

Plant-produced isoprene (2-methyl-1,3-butadiene) represents a significant portion of global volatile organic compound production, equaled only by methane. A metabolic pathway for the degradation of isoprene was first described for the Gram-positive bacterium Rhodococcus sp. AD45, and an alternative model organism has yet to be characterised. Here, we report the characterisation of a novel Gram-negative isoprene-degrading bacterium, Variovorax sp. WS11. Isoprene metabolism in this bacterium involves a plasmid-encoded iso metabolic gene cluster which differs from that found in Rhodococcus sp. AD45 in terms of organisation and regulation. Expression of iso metabolic genes is significantly upregulated by both isoprene and epoxyisoprene. The enzyme responsible for the initial oxidation of isoprene, isoprene monooxygenase, oxidises a wide range of alkene substrates in a manner which is strongly influenced by the presence of alkyl side-chains and differs from other well-characterised soluble diiron monooxygenases according to its response to alkyne inhibitors. This study presents Variovorax sp. WS11 as both a comparative and contrasting model organism for the study of isoprene metabolism in bacteria, aiding our understanding of the conservation of this biochemical pathway across diverse ecological niches.

Distinct Catalytic Behaviors between Two 1,4-Dioxane-Degrading Monooxygenases: Kinetics, Inhibition, and Substrate Range.

Monitored natural attenuation (MNA) and engineered bioremediation have been recognized as effective and cost-efficient in situ treatments to mitigate 1,4-dioxane (dioxane) contamination. Dioxane metabolism can be initiated by two catabolic enzymes, propane monooxygenase (PRM) and tetrahydrofuran monooxygenase (THM), belonging to the group-6 and 5 of soluble di-iron monooxygenase family, respectively. In this study, we comprehensively compared catalytic behaviors of PRM and THM when individually expressed in the heterologous host, Mycobacterium smegmatis mc2-155. Kinetic results revealed a half-saturation coefficient (Km) of 53.0 ± 13.1 mg/L for PRM, nearly 4 times lower than that of THM (235.8 ± 61.6 mg/L), suggesting that PRM has a higher affinity to dioxane. Exposure with three common co-contaminants (1,1-dichloroethene, trichloroethene, and 1,1,1-trichloroethane) demonstrated that PRM was also more resistant to their inhibition than THM. Thus, dioxane degraders expressing PRM may be more physiologically and ecologically advantageous than those with THM at impacted sites, where dioxane concentration is relatively low (e.g., 250 to 1000 μg/L) with co-occurrence of chlorinated solvents (e.g., 0.5 to 8 mg/L), underscoring the need of surveying both PRM and THM-encoding genes for MNA potential assessment. PRM is also highly versatile, which breaks down cyclic molecules (dioxane, tetrahydrofuran, and cyclohexane), as well as chlorinated and aromatic pollutants, including vinyl chloride, 1,2-dichloroethane, benzene, and toluene. This is the first report regarding the ability of PRM to degrade a variety of short-chain alkanes and ethene in addition to dioxane, unraveling its pivotal role in aerobic biostimulation that utilizes propane, isobutane, or other gaseous alkanes/alkenes (e.g., ethane, butane, and ethene) to select and fuel indigenous microorganisms to tackle the commingled contamination of dioxane and chlorinated compounds.

Carbon sources that enable enrichment of 1,4-dioxane-degrading bacteria in landfill leachate.

1,4-Dioxane (DX) is a recalcitrant cyclic ether that has gained attention as an emerging pollutant in the aquatic environment. Enrichment of indigenous DX-degrading bacteria, which are considered to be minor populations even in DX-impacted environments, is the key for efficient biological DX removal. Therefore, this study aimed to explore carbon sources applicable for the enrichment of DX-degrading bacteria present in landfill leachate, which is a potential source of DX pollution. Microorganisms collected from landfill leachate were cultivated on six different carbon sources (DX, tetrahydrofuran (THF), 1,3,5-trioxane (TX), ethylene glycol (EG), diethylene glycol (DEG), and 1,4-butanediol (BD)) in a sequential batch mode. Consequently, enrichment cultures cultivated on THF in addition to DX improved the DX degradation ability compared to that of the original leachate sample, while those on the other test carbon sources did not. The results indicated that THF can be an alternative carbon source to enrich DX-degrading bacteria, and that TX, EG, DEG and BD are not applicable to concentrate DX-degrading bacteria in complex microbial consortia. In addition, sequencing analyses of 16S rRNA and soluble di-iron monooxygenase (SDIMO) genes revealed notable dominance of thm/dxm genes involved in group 5 SDIMO both in DX- and THF-enrichment cultures. The analysis also showed a predominance of Pseudonocardia in THF-enrichment culture, suggesting that Pseudonocardia harboring thm/dxm genes contributes to enhanced DX degradation in THF-enrichment culture.

Enzymatic synthesis of novel water-soluble indigoid compounds

This work was aimed at expanding the diversity of indigoid compounds with novel characteristics by employing biological catalysts. A total of 16 novel indigoid compounds were synthesized using indole derivatives as substrates containing either aminomethyl-, hydroxymethyl-, carboxaldehyde or carboxyl groups at positions 4, 5, 6 or 7 of indole ring. Two different monooxygenase systems – a flavin dependent monooxygenase Hind8 and the mutant G109Q of multicomponent soluble diiron monooxygenase PML were employed to achieve the conversion of all those substrates. Characterization of purified indigo dicarboxylic acids revealed that the produced indigoids were soluble in water (solubility increased more than 1000-fold compared to indigo), also in methanol and DMSO. A bioconversion of indole-7-carboxylic acid resulted in the mixture of indigo-7,7′-dicarboxylic acid and indirubin-7,7′-dicarboxylic acid. Both of those products were separated from each another and purified thus providing the basis for the purification of water-soluble indigoids of similar structure. These findings show that (1) biological catalysts can be an easy approach for synthesizing novel indigoids with important characteristics and (2) introduction of carboxyl group can increase the water-solubility of indigo and indirubin significantly, thus providing a new tool in search for bioactive indigoids.

Stimulatory and inhibitory effects of metals on 1,4-dioxane degradation by four different 1,4-dioxane-degrading bacteria

This study evaluates the effects of various metals on 1,4-dioxane degradation by the following four bacteria: Pseudonocardia sp. D17; Pseudonocardia sp. N23; Mycobacterium sp. D6; and Rhodococcus aetherivorans JCM 14343. Eight transition metals [Co(II), Cu(II), Fe(II), Fe(III), Mn(II), Mo(VI), Ni(II), and Zn(II)] were used as the test metals. Results revealed, for the first time, that metals had not only inhibitory but also stimulatory effects on 1,4-dioxane biodegradation. Cu(II) had the most severe inhibitory effects on 1,4-dioxane degradation by all of the test strains, with significant inhibition at concentrations as low as 0.01–0.1 mg/L. This inhibition was probably caused by cellular toxicity at higher concentrations, and by inhibition of degradative enzymes at lower concentrations. In contrast, Fe(III) enhanced 1,4-dioxane degradation by Mycobacterium sp. D6 and R. aetherivorans JCM 14343 the most, while degradation by the two Pseudonocardia strains was stimulated most notably in the presence of Mn(II), even at concentrations as low as 0.001 mg/L. Enhanced degradation is likely caused by the stimulation of soluble di-iron monooxygenases (SDIMOs) involved in the initial oxidation of 1,4-dioxane. Differences in the stimulatory effects of the tested metals were likely associated with the particular SDIMO types in the test strains.

Front Cover Picture: A Biocatalytic Synthesis of Heteroaromatic N‐Oxides by Whole Cells of Escherichia coli Expressing the Multicomponent, Soluble Di‐Iron Monooxygenase (SDIMO) PmlABCDEF (Adv. Synth. Catal. 11/2019)

Front Cover Picture: A Biocatalytic Synthesis of Heteroaromatic <i>N</i>‐Oxides by Whole Cells of <i>Escherichia coli</i> Expressing the Multicomponent, Soluble Di‐Iron Monooxygenase (SDIMO) PmlABCDEF (Adv. Synth. Catal. 11/2019)

Novel tetrahydrofuran (THF) degradation-associated genes and cooperation patterns of a THF-degrading microbial community as revealed by metagenomic

Our understanding of the tetrahydrofuran (THF) degradation in complex environment is limited. The majority of THF degrading genes reported are group V soluble diiron monooxygenases and share greater than 95% homology with one another. In this study, we used sole-carbon-source incubation combined with high-throughput metagenomic sequencing to investigate this contaminant's degradation in environmental samples. We identified as-yet-uncultivated microbe from the genera Pseudonocardia and fungi Scedosporium sp. (Scedosporium sp. was successfully isolated) as THF degraders as containing THF degradation genes, while microbes from the genera Bordetella, Pandoraea and Rhodanobacter functioned as main cooperators by utilizing acidic intermediates and providing anti-acid mechanisms. Furthermore, a 9387-bp THF degradation cluster designated thmX from the as-yet-uncultivated Pseudonocardia (with 6 main ORFs and with 79–93% amino acid sequence identity with previously reported clusters) was discovered. We also found a THF-degrading related cytochrome P450 monooxygenase from the genus Scedosporium and predicted its cognate reductase for the first time. All the genes and clusters mentioned above were successfully amplified from samples and cloned into the suitable expression vectors. This study will provide novel insights for understanding of THF degradation mechanisms under acid stress conditions and mining new THF degradation genes.

Tailoring a Soluble Diiron Monooxygenase for Synthesis of Aromatic N-oxides

The aromatic N-oxides have received increased attention over the last few years due to their potential application in medicine, agriculture and organic chemistry. As a green alternative in their synthesis, the biocatalytic method employing whole cells of Escherichia coli bearing phenol monooxygenase like protein PmlABCDEF (from here on – PML monooxygenase) has been introduced. In this work, site-directed mutagenesis was used to study the contributions of active site neighboring residues I106, A113, G109, F181, F200, F209 to the regiospecificity of N-oxidation. Based on chromogenic indole oxidation screening, a collection of PML mutants with altered catalytic properties was created. Among the tested mutants, the A113G variant acquired the most distinguishable N-oxidations capacity. This new variant of PML was able to produce dioxides (quinoxaline-1,4-dioxide, 2,5-dimethylpyrazine-1,4-dioxide) and specific mono-N-oxides (2,3,5-trimethylpyrazine-1-oxide) that were unachievable using the wild type PML. This mutant also featured reshaped regioselectivity as N-oxidation shifted towards quinazoline-1-oxide compared to quinazoline-3-oxide that is produced by the wild type PML.

A Biocatalytic Synthesis of Heteroaromatic N‐Oxides by Whole Cells of Escherichia coli Expressing the Multicomponent, Soluble Di‐Iron Monooxygenase (SDIMO) PmlABCDEF

AbstractAromatic N‐oxides (ArN−OX) are desirable biologically active compounds with a potential for application in pharmacy and agriculture industries. As biocatalysis is making a great impact in organic synthesis, there is still a lack of efficient and convenient enzyme‐based techniques for the production of aromatic N‐oxides. In this study, a recombinant soluble di‐iron monooxygenase (SDIMO) PmlABCDEF overexpressed in Escherichia coli was showed to produce various aromatic N‐oxides. Out of 98 tested N‐heterocycles, seventy were converted to the corresponding N‐oxides without any side oxidation products. This whole‐cell biocatalyst showed a high activity towards pyridines, pyrazines, and pyrimidines. It was also capable of oxidizing bulky N‐heterocycles with two or even three aromatic rings. Being entirely biocatalytic, our approach provides an environmentally friendly and mild method for the production of aromatic N‐oxides avoiding the use of strong oxidants, organometallic catalysts, undesirable solvents, or other environment unfriendly reagents.magnified image

Catabolism of 2-Hydroxypyridine by Burkholderia sp. Strain MAK1: a 2-Hydroxypyridine 5-Monooxygenase Encoded by hpdABCDE Catalyzes the First Step of Biodegradation.

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.