Acidithiobacillus caldus is a chemolithoautotrophic sulfur-oxidizing bacterium that is widely used for bioleaching processes. Acidithiobacillus spp. are suggested to contain sulfur dioxygenases (SDOs) that facilitate sulfur oxidation. In this study, two putative sdo genes (A5904_0421 and A5904_1112) were detected in the genome of A. caldus MTH-04 by BLASTP searching with the previously identified SDO (A5904_0790). We cloned and expressed these genes, and detected the SDO activity of recombinant protein A5904_0421 by a GSH-dependent in vitro assay. Phylogenetic analysis indicated that A5904_0421and its homologous SDOs, mainly found in autotrophic bacteria, were distantly related to known SDOs and were categorized as a new subgroup of SDOs. The potential functions of genes A5904_0421 (termed sdo1) and A5904_0790 (termed sdo2) were investigated by generating three knockout mutants (Δsdo1, Δsdo2 and Δsdo1&2), two sdo overexpression strains (OE-sdo1 and OE-sdo2) and two sdo complemented strains (Δsdo1/sdo1' and Δsdo2/sdo2') of A. caldus MTH-04. Deletion or overexpression of the sdo genes did not obviously affect growth of the bacteria on S0, indicating that the SDOs did not play an essential role in the oxidation of extracellular elemental sulfur in A. caldus. The deletion of sdo1 resulted in complete inhibition of growth on tetrathionate, slight inhibition of growth on thiosulfate and increased GSH-dependent sulfur oxidation activity on S0. Transcriptional analysis revealed a strong correlation between sdo1 and the tetrathionate intermediate pathway. The deletion of sdo2 promoted bacterial growth on tetrathionate and thiosulfate, and overexpression of sdo2 altered gene expression patterns of sulfide:quinone oxidoreductase and rhodanese. Taken together, the results suggest that sdo1 is essential for the survival of A. caldus when tetrathionate is used as the sole energy resource, and sdo2 may also play a role in sulfur metabolism.

Epitranscriptomic RNA modifications can regulate fundamental biological processes, but we lack approaches to map modification sites and probe writer enzymes. Here we present a chemoproteomic strategy to characterize RNA 5-methylcytidine (m5C) dioxygenase enzymes in their native context based upon metabolic labeling and activity-based crosslinking with 5-ethynylcytidine (5-EC). We profile m5C dioxygenases in human cells including ALKBH1 and TET2 and show that ALKBH1 is the major hm5C- and f5C-forming enzyme in RNA. Further, we map ALKBH1 modification sites transcriptome-wide using 5-EC-iCLIP and ARP-based sequencing to identify ALKBH1-dependent m5C oxidation in a variety of tRNAs and mRNAs and analyze ALKBH1 substrate specificity in vitro. We also apply targeted pyridine borane-mediated sequencing to measure f5C sites on select tRNA. Finally, we show that f5C at the wobble position of tRNA-Leu-CAA plays a role in decoding Leu codons under stress. Our work provides powerful chemical approaches for studying RNA m5C dioxygenases and mapping oxidative m5C modifications and reveals the existence of novel epitranscriptomic pathways for regulating RNA function.

Oxygenases are ubiquitous enzymes that catalyze the introduction of one or two oxygen atoms to unreactive chemical compounds. They require reduction equivalents from NADH or NADPH and comprise metal ions, metal ion complexes, or coenzymes in their active site. Thus, for industrial purposes, oxygenases are most commonly employed using whole cell catalysis, to alleviate the need for co-factor regeneration. Biotechnological applications include bioremediation, chiral synthesis, biosensors, fine chemicals, biofuels, pharmaceuticals, food ingredients and polymers. Controlling activity and selectivity of oxygenases is therefore of great importance and of growing interest to the scientific community. This review focuses on protein engineering of non-heme monooxygenases and dioxygenases for generating improved or novel functionalities. Rational mutagenesis based on x-ray structures and sequence alignment, as well as random methods such as directed evolution, have been utilized. It is concluded that knowledge-based protein engineering accompanied with targeted libraries, is most efficient for the design and tuning of biocatalysts towards novel substrates and enhanced catalytic activity while minimizing the screening efforts.

α-Dioxygenases (α-DOXs) are known as plant enzymes involved in the α-oxidation of fatty acids through which fatty aldehydes, with a high commercial value as flavor and fragrance compounds, are synthesized as products. Currently, little is known about α-DOXs from non-plant organisms. The phylogenic analysis reported here identified a substantial number of α-DOX enzymes across various taxa. Here, we report the functional characterization and Escherichia coli whole-cell application of two novel α-DOXs identified from cyanobacteria: CalDOX from Calothrix parietina and LepDOX from Leptolyngbya sp. The catalytic behavior of the recombinantly expressed CalDOX and LepDOX revealed that they are heme-dependent like plant α-DOXs but exhibit activities toward medium carbon fatty acids ranging from C10 to C14 unlike plant α-DOXs. The in-depth molecular investigation of cyanobacterial α-DOXs and their application in an E. coli whole system employed in this study is useful not only for the understanding of the molecular function of α-DOXs, but also for their industrial utilization in fatty aldehyde biosynthesis.Key points• Two novel α-dioxygenases from Cyanobacteria are reported• Both enzymes prefer medium-chain fatty acids• Both enzymes are useful for fatty aldehyde biosynthesis.

Coumarins are natural plant products that have been the subject of extensive phytochemical and pharmacological research studies in the past few decades. The core structure of coumarins is derived from the respective cinnamates via ortho-hydroxylation of the aromatic ring, trans/cis isomerization, and lactonization. Various substitution patterns of coumarins have been reported, whereas the biosynthesis of coumarins remains elusive. Ortho-hydroxylation is a key step in simple coumarin biosynthesis as a branch point from the lignin biosynthetic pathway. 2-Oxoglutarate-dependent dioxygenases (2OGDs) from plants convert cinnamate derivatives into simple coumarins through the process of ortho-hydroxylation. This review describes the 2OGDs involved in coumarin biosynthesis and their substrate specificities.

Abundant evidence connects depression symptomology with immune system activation, stress and subsequently elevated levels of kynurenine. Anti-depressants, such as the tricyclic norepinephrine/serotonin reuptake inhibitor desipramine (Desip), were developed under the premise that increasing extracellular neurotransmitter level was the sole mechanism by which they alleviate depressive symptomologies. However, evidence suggests that anti-depressants have additional actions that contribute to their therapeutic potential. The Kynurenine Pathway produces tryptophan metabolites that modulate neurotransmitter activity. This recognition identified another putative pathway for anti-depressant targeting. Considering a recognized role of the Kynurenine Pathway in depression, we investigated the potential for Desip to alter expression of rate-limiting enzymes of this pathway: indoleamine-2,3-dioxygenases (Ido1 and Ido2). Mice were administered lipopolysaccharide (LPS) or synthetic glucocorticoid dexamethasone (Dex) with Desip to determine if Desip alters indoleamine-dioxygenase (DO) expression in vivo following a modeled immune and stress response. This work was followed by treating murine and human peripheral blood mononuclear cells (PBMCs) with interferon-gamma (IFNγ) and Desip. In vivo: Desip blocked LPS-induced Ido1 expression in hippocampi, astrocytes, microglia and PBMCs and Ido2 expression by PBMCs. Ex vivo: Desip decreased IFNγ-induced Ido1 and Ido2 expression in murine PBMCs. This effect was directly translatable to the human system as Desip decreased IDO1 and IDO2 expression by human PBMCs. These data demonstrate for the first time that an anti-depressant alters expression of Ido1 and Ido2, identifying a possible new mechanism behind anti-depressant activity. Furthermore, we propose the assessment of PBMCs for anti-depressant responsiveness using IDO expression as a biomarker.

Role of microbes in bioremediation of oil spills has become inevitable owing to their eco friendly nature. This study focused on the isolation and characterization of bacterial strains with superior oil degrading potential from crude-oil contaminated soil. Three such bacterial strains were selected and subsequently identified by 16S rRNA gene sequence analysis as Corynebacterium aurimucosum, Acinetobacter baumannii and Microbacterium hydrocarbonoxydans respectively. The specific activity of catechol 1,2 dioxygenase (C12O) and catechol 2,3 dioxygenase (C23O) was determined in these three strains wherein the activity of C12O was more than that of C23O. Among the three strains, Microbacterium hydrocarbonoxydans exhibited superior crude oil degrading ability as evidenced by its superior growth rate in crude oil enriched medium and enhanced activity of dioxygenases. Also degradation of total petroleum hydrocarbon (TPH) in crude oil was higher with Microbacterium hydrocarbonoxydans. The three strains also produced biosurfactants of glycolipid nature as indicated d by biochemical, FTIR and GCMS analysis. These findings emphasize that such bacterial strains with superior oil degrading capacity may find their potential application in bioremediation of oil spills and conservation of marine and soil ecosystem.

Carotenoid cleavage dioxygenases (CCDs) are a group of enzymes that catalyze the oxidative cleavage steps from carotenoids to various carotenoid cleavage products. Some ccd genes have been identified and encoded enzymes functionally characterized in many higher plants, but little in cyanobacteria. We performed a comparative analysis of ccd sequences and explored their distribution, classification, phylogeny, evolution, and structure among 37 cyanobacteria. Totally 61 putative ccd sequences were identified, which are abundant in Acaryochloris marina MBIC 11017, filamentous N(2)-fixing cyanobacteria, and unicellular cyanobacterial Cyanothece. According to phylogenetic trees of 16S rDNA and CCD, nced and ccd8 genes occur later than the divergence of ccd7, apco, and ccd1. All CCD enzymes share conserved basic structure domains constituted by a single loop formed with seven β-strands and one helix. In this paper, a general framework of sequence-function-evolution connection for the ccd has been revealed, which may provide new insight for functional investigation.

The 2-oxoglutarate dependent superfamily is a diverse group of non-haem dioxygenases, and is present in prokaryotes, eukaryotes, and archaea. The enzymes differ in substrate preference and reaction chemistry, a factor that precludes their classification by homology studies and electronic annotation schemes alone. In this work, I propose and explore the rationale of using substrates to classify structurally similar alpha-ketoglutarate dependent enzymes.

L-Ascorbic acid (ascorbate, Vitamin C) is an essential human micronutrient that is predominantly obtained from plants. It is known to work as the major antioxidant in plants, and it underpins several environmentally induced stresses due to its use as a co-factor by certain 2-oxoglutarate-dependent (2-OG) dioxygenases [2(OG)-dioxygenases]. It is important to understand the role of 2(OG)-dioxygenases in the biosynthesis of ascorbate. The present study examined contents of ascorbate and protein-protein interaction in nine T-DNA mutants of Arabidopsis containing an insert in their respective (2-OG) dioxygenase genes (At1g20270, At1g68080, At2g17720, At3g06290, At3g28490, At4g35810, At4g35820, At5g18900, At5g66060). In this study, the amount of ascorbate in five of the mutants was shown to be almost two-fold or more than two-fold higher than in the wild type. This result may be a consequence of the insertion of the T-DNA. The prediction of possible protein interactions between 2(OG)-dioxygenases and relevant ascorbate-function players may indicate the oxidative effects of certain dioxygenase proteins in plants. It is expected that certain dioxygenases are actively involved in the metabolic and biosynthetic pathways of ascorbate. This involvement may be of importance to increase ascorbate amounts in plants for human nutrition, and to protect plant species against stress conditions.

Iron(II) and 2-oxoglutarate (2OG)-dependent dioxygenases involved in histone and DNA/RNA demethylation convert the cosubstrate 2OG and oxygen to succinate and carbon dioxide, resulting in hydroxylation of the methyl group of the substrates and subsequent demethylation. Recent evidence has shown that these 2OG dioxygenases play vital roles in a variety of biological processes, including transcriptional regulation and gene expression. In this review, the structure and function of these dioxygenases in histone and nucleic acid demethylation will be discussed. Given the important roles of these 2OG dioxygenases, detailed analysis and comparison of the 2OG dioxygenases will guide the design of target-specific small-molecule chemical probes and inhibitors.

DNA methylation at the C-5 position of cytosine (5mC) regulates gene expression and plays pivotal roles in various biological processes. The TET dioxygenases catalyze iterative oxidation of 5mC, leading to eventual demethylation. Inactivation of TET enzymes causes multistage developmental defects, impaired cell reprogramming, and hematopoietic malignancies. However, little is known about how TET activity is regulated. Here we show that all three TET proteins bind to VprBP and are monoubiquitylated by the VprBP-DDB1-CUL4-ROC1 E3 ubiquitin ligase (CRL4(VprBP)) on a highly conserved lysine residue. Deletion of VprBP in oocytes abrogated paternal DNA hydroxymethylation in zygotes. VprBP-mediated monoubiquitylation promotes TET binding to chromatin. Multiple recurrent TET2-inactivating mutations derived from leukemia target either the monoubiquitylation site (K1299) or residues essential for VprBP binding. Cumulatively, our data demonstrate that CRL4(VprBP) is a critical regulator of TET dioxygenases during development and in tumor suppression.

Lignostilbene-α,β-dioxygenases (LSDs) are iron-dependent oxygenases involved in the catabolism of lignin-derived stilbenes. Sphingobium sp. SYK-6 contains eight LSD homologs with undetermined physiological roles. To investigate which homologs are involved in the catabolism of dehydrodiconiferyl alcohol (DCA), derived from β-5 linked lignin subunits, we heterologously produced the enzymes and screened their activities in lysates. The seven soluble enzymes all cleaved lignostilbene, but only LSD2, LSD3, and LSD4 exhibited high specific activity for 3-(4-hydroxy-3-(4-hydroxy-3-methoxystyryl)-5-methoxyphenyl) acrylate (DCA-S) relative to lignostilbene. LSD4 catalyzed the cleavage of DCA-S to 5-formylferulate and vanillin and cleaved lignostilbene and DCA-S (∼106 M-1 s-1) with tenfold greater specificity than pterostilbene and resveratrol. X-ray crystal structures of native LSD4 and the catalytically inactive cobalt-substituted Co-LSD4 at 1.45 Å resolution revealed the same fold, metal ion coordination, and edge-to-edge dimeric structure as observed in related enzymes. Key catalytic residues, Phe-59, Tyr-101, and Lys-134, were also conserved. Structures of Co-LSD4·vanillin, Co-LSD4·lignostilbene, and Co-LSD4·DCA-S complexes revealed that Ser-283 forms a hydrogen bond with the hydroxyl group of the ferulyl portion of DCA-S. This residue is conserved in LSD2 and LSD4 but is alanine in LSD3. Substitution of Ser-283 with Ala minimally affected the specificity of LSD4 for either lignostilbene or DCA-S. By contrast, substitution with phenylalanine, as occurs in LSD5 and LSD6, reduced the specificity of the enzyme for both substrates by an order of magnitude. This study expands our understanding of an LSD critical to DCA catabolism as well as the physiological roles of other LSDs and their determinants of substrate specificity.

Oxidative enzymes catalyze many different reactions in plant metabolism. Among this suite of enzymes are the 2-oxoglutarate/Fe(II)-dependent dioxygenases (2-ODDs). Cytochromes P450 (CYPs) as often considered the most versatile oxidative enzymes in nature, but the diversity and complexity of reactions catalyzed by 2-ODDs is superior to the CYPs. The list of oxidative reactions catalyzed by 2-ODDs includes hydroxylations, demethylations, desaturations, ring closure, ring cleavage, epimerization, rearrangement, halogenation, and demethylenation. Furthermore, recent work, including the discovery of 2-ODDs involved in epigenetic regulation, and others catalyzing several characteristic steps in specialized metabolic pathways, support the argument that 2-ODDs are among the most versatile and important oxidizing biological catalysts. In this review, we survey and summarize the pertinent literature with a focus on several key reactions catalyzed by 2-ODDs, and discuss the significance and impact of these enzymes in plant metabolism.

Ten-eleven translocation (TET) enzymes are implicated in DNA demethylation through dioxygenase activity, which converts 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC). However, the specific roles of TET enzymes and 5-hmC levels in head and neck squamous cell carcinoma (HNSCC) have not yet been evaluated. In this study, we analyzed 5-hmC levels and TET mRNA expression in a well-characterized dataset of 117 matched pairs of HNSCC tissues and normal tissues. 5-hmC levels and TET mRNA expression were examined via enzyme-linked immunosorbent assay and quantitative real-time PCR, respectively. 5-hmC levels were evaluated according to various clinical characteristics and prognostic implications. Notably, we found that 5-hmC levels were significantly correlated with tumor stage (P = 0.032) and recurrence (P = 0.018). Univariate analysis revealed that low levels of 5-hmC were correlated with poor disease-free survival (DFS; log-rank test, P = 0.038). The expression of TET family genes was not associated with outcomes. In multivariate analysis, low levels of 5-hmC were evaluated as a significant independent prognostic factor of DFS (hazard ratio: 2.352, 95% confidence interval: 1.136-4.896; P = 0.021). Taken together, our findings showed that reduction of TET family gene expression and subsequent low levels of 5-hmC may affect the development of HNSCC.

It is clear that 2-oxoglutarate-dependent dioxygenases have critical functions in salicylic acid (SA) metabolism in plants, yet their role in SA biosynthesis is poorly understood. Here, we report that two dioxygenase-encoding genes, SLENDER AND CRINKLY LEAF1 (SLC1) and SLC2, play essential roles in shoot development and SA production in rice. Overexpression of SLC1 (SLC1-OE) or SLC2 (SLC2-OE) in rice produced infertile plants with slender and crinkly leaves. Disruption of SLC1 or SLC2 led to dwarf plants, while simultaneous down-regulation of SLC1 and SLC2 resulted in a severe defect in early leaf development. Enhanced SA levels in SLC1-OE plants and decreased SA levels in slc1 and slc2 mutants were observed. Accordingly, these lines all showed altered expression of a set of SA-related genes. We demonstrated that SLC1 interacts with homeobox1 (OSH1), and that either the knotted1-like homeobox (KNOX1) or glutamate, leucine, and lysine (ELK) domain of OSH1 is sufficient for accomplishing this interaction. Collectively, our data reveal the importance of SLC1 and SLC2 in rice shoot development.

TET/JBP enzymes oxidize 5-methylpyrimidines in DNA. In mammals, the oxidized methylcytosines (oxi-mCs) function as epigenetic marks and likely intermediates in DNA demethylation. Here we present a method based on diglucosylation of 5-hydroxymethylcytosine (5hmC) to simultaneously map 5hmC, 5-formylcytosine, and 5-carboxylcytosine at near-base-pair resolution. We have used the method to map the distribution of oxi-mC across the genome of Coprinopsis cinerea, a basidiomycete that encodes 47 TET/JBP paralogs in a previously unidentified class of DNA transposons. Like 5-methylcytosine residues from which they are derived, oxi-mC modifications are enriched at centromeres, TET/JBP transposons, and multicopy paralogous genes that are not expressed, but rarely mark genes whose expression changes between two developmental stages. Our study provides evidence for the emergence of an epigenetic regulatory system through recruitment of selfish elements in a eukaryotic lineage, and describes a method to map all three different species of oxi-mCs simultaneously.

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered report describes the proposed replication plan of key experiments from 'Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of α-ketoglutarate-dependent dioxygenases' by Xu and colleagues, published in Cancer Cell in 2011 (Xu et al., 2011). The key experiments being replicated include Supplemental Figure 3I, which demonstrates that transfection with mutant forms of IDH1 increases levels of 2-hydroxyglutarate (2-HG), Figures 3A and 8A, which demonstrate changes in histone methylation after treatment with 2-HG, and Figures 3D and 7B, which show that mutant IDH1 can effect the same changes as treatment with excess 2-HG. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife.

The iron(II)- and 2-oxoglutarate (2OG)-dependent dioxygenase AlkB from Escherichia coli (EcAlkB) repairs alkylation damage in DNA by direct reversal. EcAlkB substrates include methylated bases, such as 1-methyladenine (m(1)A) and 3-methylcytosine (m(3)C), as well as certain bulkier lesions, for example the exocyclic adduct 1,N(6)-ethenoadenine (epsilonA). EcAlkB is the only bacterial AlkB protein characterized to date, and we here present an extensive bioinformatics and functional analysis of bacterial AlkB proteins. Based on sequence phylogeny, we show that these proteins can be subdivided into four groups: denoted 1A, 1B, 2A and 2B; each characterized by the presence of specific conserved amino acid residues in the putative nucleotide-recognizing domain. A scattered distribution of AlkB proteins from the four different groups across the bacterial kingdom indicates a substantial degree of horizontal transfer of AlkB genes. DNA repair activity was associated with all tested recombinant AlkB proteins. Notably, both a group 2B protein from Xanthomonas campestris and a group 2A protein from Rhizobium etli repaired etheno adducts, but had negligible activity on methylated bases. Our data indicate that the majority, if not all, of the bacterial AlkB proteins are DNA repair enzymes, and that some of these proteins do not primarily target methylated bases.

Carotenoid cleavage dioxygenases (CCDs) selectively catalyze carotenoids, forming smaller apocarotenoids that are essential for the synthesis of apocarotenoid flavor, aroma volatiles, and phytohormone ABA/SLs, as well as responses to abiotic stresses. Here, 19, 11, and 10 CCD genes were identified in Nicotiana tabacum, Nicotiana tomentosiformis, and Nicotiana sylvestris, respectively. For this family, we systematically analyzed phylogeny, gene structure, conserved motifs, gene duplications, cis-elements, subcellular and chromosomal localization, miRNA-target sites, expression patterns with different treatments, and molecular evolution. CCD genes were classified into two subfamilies and nine groups. Gene structures, motifs, and tertiary structures showed similarities within the same groups. Subcellular localization analysis predicted that CCD family genes are cytoplasmic and plastid-localized, which was confirmed experimentally. Evolutionary analysis showed that purifying selection dominated the evolution of these genes. Meanwhile, seven positive sites were identified on the ancestor branch of the tobacco CCD subfamily. Cis-regulatory elements of the CCD promoters were mainly involved in light-responsiveness, hormone treatment, and physiological stress. Different CCD family genes were predominantly expressed separately in roots, flowers, seeds, and leaves and exhibited divergent expression patterns with different hormones (ABA, MeJA, IAA, SA) and abiotic (drought, cold, heat) stresses. This study provides a comprehensive overview of the NtCCD gene family and a foundation for future functional characterization of individual genes.