Acid Dioxygenase Research Articles

Ease of sutureless aortic valve replacement in a patient with unexpected ochronosis: a case report

BackgroundAlkaptonuria is a rare congenital metabolic disorder characterized by homogentisic acid accumulation in body cartilage and connective tissues due to a deficient homogentisic acid dioxygenase enzyme. This disorder manifests in various clinical symptoms, including spondyloarthropathy, ocular and dermal pigmentation, genitourinary tract obstruction by ochronosis stones, and cardiovascular system involvement. Cardiac ochronosis is a rare manifestation of alkaptonuria that may present as aortic stenosis, sometimes accompanied by other cardiovascular complications.Case presentationWe report an unexpected case of ochronosis diagnosed during cardiac surgery. Due to the fragile, thin, and atheromatous nature of the ascending aorta in patients with ochronosis, we opted for a sutureless aortic valve replacement procedure. This approach appears to be more suitable for patients with ochronosis.ConclusionsAlthough cardiac ochronosis is rare, surgeons should remain vigilant and consider the possibility of this condition when examining patients with aortic valve stenosis, paying close attention to the clinical manifestations of alkaptonuria.

On the benefits of the tryptophan metabolite 3-hydroxyanthranilic acid in Caenorhabditis elegans and mouse aging

Tryptophan metabolism through the kynurenine pathway influences molecular processes critical to healthy aging including immune signaling, redox homeostasis, and energy production. Aberrant kynurenine metabolism occurs during normal aging and is implicated in many age-associated pathologies including chronic inflammation, atherosclerosis, neurodegeneration, and cancer. We and others previously identified three kynurenine pathway genes—tdo-2, kynu-1, and acsd-1—for which decreasing expression extends lifespan in invertebrates. Here we report that knockdown of haao-1, a fourth gene encoding the enzyme 3-hydroxyanthranilic acid (3HAA) dioxygenase (HAAO), extends lifespan by ~30% and delays age-associated health decline in Caenorhabditis elegans. Lifespan extension is mediated by increased physiological levels of the HAAO substrate 3HAA. 3HAA increases oxidative stress resistance and activates the Nrf2/SKN-1 oxidative stress response. In pilot studies, female Haao knockout mice or aging wild type male mice fed 3HAA supplemented diet were also long-lived. HAAO and 3HAA represent potential therapeutic targets for aging and age-associated disease.

Herbicide resistance in Bromus spp.: a global review

AbstractThis review summarizes what is currently known about herbicide resistance in Bromus spp. worldwide. Additional information on the biology and genetics of Bromus spp. is provided to further the understanding of resistance evolution and dispersal of the different species. Cases of herbicide resistance have been confirmed in Bromus catharticus Vahl., Bromus commutatus Schrad. (syn.: Bromus racemosus L.), Bromus diandrus Roth, Bromus japonicus Thunb. (syn.: Bromus arvensis L.), Bromus madritensis L., Bromus rigidus Roth (syn.: Bromus diandrus Roth ssp. diandrus), Bromus rubens L., Bromus secalinus L., Bromus sterilis L., and Bromus tectorum L. in 11 countries. Bromus spp. populations have evolved cross- and multiple resistance to six herbicide sites of action: acetyl-coenzyme A carboxylase, acetolactate synthase, photosystem II, very-long-chain fatty-acid, 5-enolpyruvylshikimate-3-phosphate synthase, and 4-hydroxyphenylpyruvate dioxygenase inhibitors. Resistance mechanisms varied from target-site to non–target site or a combination of both. Bromus spp. are generally highly self-pollinated, but outcrossing can occur at low levels in some species. Bromus spp. have different ploidy levels, ranging from diploid (2n = 2x = 14) to duodecaploid (2n = 12x = 84). Herbicide resistance in Bromus spp. is a global issue, and the spread of herbicide-resistance alleles primarily occurs via seed-mediated gene flow. However, the transfer of herbicide-resistance alleles via pollen-mediated gene flow is possible.

Cyanide replaces substrate in obligate-ordered addition of nitric oxide to the non-heme mononuclear iron AvMDO active site.

Thiol dioxygenases are a subset of non-heme mononuclear iron oxygenases that catalyze the O2-dependent oxidation of thiol-bearing substrates to yield sulfinic acid products. Cysteine dioxygenase (CDO) and 3-mercaptopropionic acid (3MPA) dioxygenase (MDO) are the most extensively characterized members of this enzyme family. As with many non-heme mononuclear iron oxidase/oxygenases, CDO and MDO exhibit an obligate-ordered addition of organic substrate before dioxygen. As this substrate-gated O2-reactivity extends to the oxygen-surrogate, nitric oxide (NO), EPR spectroscopy has long been used to interrogate the [substrate:NO:enzyme] ternary complex. In principle, these studies can be extrapolated to provide information about transient iron-oxo intermediates produced during catalytic turnover with dioxygen. In this work, we demonstrate that cyanide mimics the native thiol-substrate in ordered-addition experiments with MDO cloned from Azotobacter vinelandii (AvMDO). Following treatment of the catalytically active Fe(II)-AvMDO with excess cyanide, addition of NO yields a low-spin (S = 1/2) (CN/NO)-Fe-complex. Continuous wave and pulsed X-band EPR characterization of this complex produced in wild-type and H157N variant AvMDO reveal multiple nuclear hyperfine features diagnostic of interactions within the first- and outer-coordination sphere of the enzymatic Fe-site. Spectroscopically validated computational models indicate simultaneous coordination of two cyanide ligands replaces the bidentate (thiol and carboxylate) coordination of 3MPA allowing for NO-binding at the catalytically relevant O2-binding site. This promiscuous substrate-gated reactivity of AvMDO with NO provides an instructive counterpoint to the high substrate-specificity exhibited by mammalian CDO for L-cysteine.

EXTENDING A HEALTHY LIFESPAN WITH 3-HYDROXYANTHRANILIC ACID

Abstract Metabolism of tryptophan by the kynurenine pathway is increasingly linked to aging. Kynurenine pathway enzymes and metabolites influence a range of molecular processes critical to healthy aging, including regulation of inflammatory and immune responses, cellular redox homeostasis, and energy production. Aberrant kynurenine metabolism occurs during normal aging and is implicated in many age-associated pathologies including chronic inflammation, atherosclerosis, neurodegeneration, and cancer. We and others previously identified three kynurenine pathway genes—kynu-1, tdo-2, and acsd-1—for which decreasing expression extends lifespan in invertebrates. More recently we discovered that knockdown of haao-1, a fourth kynurenine pathway gene encoding the enzyme 3-hydroxyanthranilic acid dioxygenase (HAAO), extends lifespan by ~30% and delays age-associated health decline in Caenorhabditis elegans. Lifespan extension is mediated by increased physiological levels of the HAAO substrate 3-hydroxyanthranilic acid (3HAA). Aging mice fed a diet supplemented with 3HAA are similarly long-lived. The mechanism of action liking 3HAA to aging is complex and partially overlaps with multiple pathways previously implicated in aging. We recently identified activation of the Nrf2/SKN-1 oxidative stress response and alterations to iron homeostasis as key players in the benefits 3HAA. Ongoing work explores the relationship between 3HAA, Nrf2/SKN-1, and iron in C. elegans and mammalian aging, age-associated immune decline, and cancer. This works provides a foundation for detailed examination of the molecular mechanisms underlying the benefits of 3HAA, and how these mechanisms interact with other anti-aging interventions. We anticipate that these findings will bolster growing interest in developing pharmacological strategies to target tryptophan metabolism to improve health aging.

The metabolites of de novo NAD+ synthesis are a valuable predictor of acute kidney injury.

Acute kidney injury (AKI) is often iatrogenic and potentially preventable. Reduced renal nicotinamide adenine dinucleotide (NAD+) is reported to increase the susceptibility of AKI. The present study explored the predictive value of urinary de novo NAD+ synthetic metabolites for AKI using two independent cohorts. The expression of de novo NAD+ synthetic enzymes in human kidney was examined by immunohistochemistry and single-cell transcriptomes. Urine samples were collected from two independent cohorts: the methotrexate (MTX) cohort with high-dose MTX treatment for lymphoma (n=189) and the liver transplantation cohort with orthotopic liver transplantation (n=49). Urinary metabolomics study of NAD+ de novo synthesis was performed by liquid chromatography with mass spectrometry, screening for AKI predictive biomarkers. Nephroseq database and immunohistochemistry were used to analyze kidney de novo NAD+ synthetic enzymes expression in AKI-susceptible conditions. Human proximal tubule was the main structure in the kidney that expressed the necessary enzymes for NAD+ de novo synthesis. In the MTX cohort, the urinary quinolinic acid (QA)/3-hydroxyanthranilic acid (3-OH AA) ratio before chemotherapy was significantly lower in those who developed AKI after chemotherapy compared with those who did not. This finding was consistent in the liver transplantation cohort. The area under the receiver-operating characteristic curve (AUC) of urinary QA/3-OH AA for AKI prediction was 0.749 and 0.729 in two cohorts, respectively. 3-Hydroxyanthranilic acid dioxygenase (HAAO), the enzyme catalyzing QA synthesis from 3-OH AA, decreased in AKI-susceptible diabetic kidneys. The human proximal tubules were important source of NAD+ from the de novo pathway. Reduced urinary QA/3-OH AA ratio, which possibly suggested decreased HAAO activity, could be a potential AKI predictive biomarker.

The Effect of an α-7 Nicotinic Allosteric Modulator PNU120596 and NMDA Receptor Antagonist Memantine on Depressive-like Behavior Induced by LPS in Mice: The Involvement of Brain Microglia.

Nicotinic acetylcholine receptors (nAChRs), particularly the α7 nAChR, play a critical role in neuroinflammation and microglial activation associated with major depressive disorder (MDD). Microglial quinolinic acid (QUIN), which is synthesized by 3-hydroxyanthranilic acid dioxygenase (HAAO), is an N-methyl-D-aspartate (NMDA) receptor agonist and has been implicated in the development of MDD-related symptoms. In the present study, we assessed the effects of PNU120596, an α7 nAChR positive allosteric modulator (PAM), on HAAO expression and QUIN formation in the hippocampus and prefrontal cortex. We also investigated the effects of memantine, an NMDA receptor antagonist, alone and in combination with PNU120596 on cognitive deficit and depressive-like behaviors induced by lipopolysaccharide (LPS) in mice using the Y-maze and forced swim test, respectively. LPS (1 mg/kg, i.p.) elevated HAAO expression and QUIN formation in the hippocampus and prefrontal cortex, which were reduced with pretreatment with PNU120596 (4 mg/kg, i.p.). Furthermore, memantine (1 or 3 mg/kg, i.p.) prevented the cognitive deficit and depressive-like behaviors induced by LPS in mice. Together, these results suggest that the antidepressant-like effects of PNU120596 are mediated by attenuation of LPS-induced QUIN formation. Therefore, α7 nAChR PAM could be a potential therapeutic candidate for MDD associated with neurotoxic glutamatergic transmission.

Transcriptomic analyses reveal increased expression of dioxygenases, monooxygenases, and other metabolizing enzymes involved in anthracene degradation in the marine alga Ulva lactuca.

To analyze the mechanisms involved in anthracene (ANT) degradation in the marine alga Ulva lactuca, total RNA was obtained from the alga cultivated without ANT and with 5 μM of ANT for 24 h, and transcriptomic analyses were performed. A de novo transcriptome was assembled, transcripts differentially expressed were selected, and those overexpressed were identified. Overexpressed transcripts potentially involved in ANT degradation were: one aromatic ring dioxygenase, three 2-oxoglutarate Fe (II) dioxygenases (2-OGDOs), and three dienelactone hydrolases that may account for anthraquinone, phthalic anhydride, salicylic acid, and phthalic acid production (pathway 1). In addition, two flavin adenine dinucleotide (FAD)-dependent monooxygenases, four cytP450 monooxygenases, two epoxide hydrolase, one hydroxyphenylpyruvic acid dioxygenase (HPPDO), and two homogentisic acid dioxygenases (HGDOs) were identified that may also participate in ANT degradation (pathway 2). Moreover, an alkane monooxygenase (alkB), two alcohol dehydrogenases, and three aldehyde dehydrogenases were identified, which may participate in linear hydrocarbon degradation (pathway 3). Furthermore, the level of transcripts encoding some of mentioned enzymes were quantified by qRT-PCR are in the alga cultivated with 5 μM of ANT for 0–48 h, and those more increased were 2-OGDO, HGDO, and alkB monooxygenase. Thus, at least three pathways for ANT and linear hydrocarbons degradation may be existed in U. lactuca. In addition, ANT metabolites were analyzed by gas chromatography and mass spectrometry (GC–MS), allowing the identification of anthraquinone, phthalic anhydride, salicylic acid, and phthalic acid, thus validating the pathway 1.

The Biosynthesis of 1-octene-3-ol by a Multifunctional Fatty Acid Dioxygenase and Hydroperoxide Lyase in Agaricus bisporus.

The biosynthetic pathway from linoleic acid to 1-octen-3-ol in Agaricus bisporus has long been established, in which linoleic acid is converted to 10-hydroperoxide (10-HPOD) by deoxygenation, and 10-HPOD is subsequently cleaved to yield 1-octene-3-ol and 10-oxodecanoic acid. However, the corresponding enzymes have not been identified and cloned. In the present study, four putative genes involved in oxylipid biosynthesis, including one lipoxygenase gene named AbLOX, two linoleate diol synthase genes named AbLDS1 and AbLDS2, and one hydroperoxide lyase gene named AbHPL were retrieved from the A. bisporus genome by a homology search and cloned and expressed prokaryotically. AbLOX, AbLDS1, and AbLDS2 all exhibited fatty acid dioxygenase activity, catalyzing the conversion of linoleic acid to generate hydroperoxide, and AbHPL showed a cleaving hydroperoxide activity, as was determined by the KI-starch method. AbLOX and AbHPL catalyzed linoleic acid to 1-octen-3-ol with an optimum temperature of 35 °C and an optimum pH of 7.2, whereas AbLDS1, AbLDS2, and AbHPL catalyzed linoleic acid without 1-octen-3-ol. Reduced AbLOX expression in antisense AbLOX transformants was correlated with a decrease in the yield of 1-octen-3-ol. AbLOX and AbHPL were highly homologous to the sesquiterpene synthase Cop4 of Coprinus cinerea and the yeast sterol C-22 desaturase, respectively. These results reveal that the enzymes for the oxidative cleavage of linoleic acid to synthesize 1-octen-3-ol in A. bisporus are the multifunctional fatty acid dioxygenase AbLOX and hydroperoxide lyase AbHPL.

Whole genome sequencing and analysis of fenvalerate degrading bacteria Citrobacter freundii CD-9.

Citrobacter freundii CD-9 is a Gram-negative bacteria sourced from factory sludge that can use fenvalerate as its sole carbon source and has a broad degradation spectrum for pyrethroid pesticides. The whole genome of CD-9 sequenced using Illumina HiSeq PE150 was reported in this study. The CD-9 genome size was 5.33 Mb and the G + C content was 51.55%. A total of 5291 coding genes, 9 5s-rRNA, and 79 tRNA were predicted bioinformatically. 3586 genes annotated to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database that can be involved in 173 metabolic pathways, including various microbial metabolic pathways that degrade exogenous chemicals, especially those that degrade aromatic compounds, and also produce a variety of bioactive substances. Fifty genes related to pyrethroid degradation were identified in the C. freundii CD-9 genome, including 9 dioxygenase, 25 hydrolase, and 16 esterase genes. Notably, RT-qPCR results showed that from the predicted 13 genes related to fenvalerate degradation, the expression of six genes, including esterase, HAD family hydrolase, lipolytic enzyme, and gentisic acid dioxygenase, was induced in the presence of fenvalerate. In this study, the key genes and degradation mechanism of C. freundii CD-9 were analyzed and the results provide scientific evidence to support its application in environmental bioremediation. It can establish application models for different environmental pollution management by constructing genetically engineered bacteria for efficient fenvalerate or developing enzyme formulations that can be industrially produced.

Kynurenine Metabolism Lifespan Extension Mediated by Oxidative Stress Response and Hypoxic Response in C. elegans

Abstract Aging is characterized by a progressive decline in the normal physiological functions of an organism, ultimately leading to mortality. Metabolic changes throughout the aging process disrupt the balance and homeostasis of the cell. The kynurenine metabolic pathway is the sole de novo biosynthetic pathway for producing NAD+ from ingested tryptophan. Altered kynurenine pathway activity is associated with both aging and a variety of age-associated diseases, and kynurenine-based interventions can extend lifespan in Caenorhabditis elegans. Our laboratory recently demonstrated knockdown of the kynurenine pathway enzymes kynureninase (KYNU) or 3-hydroxyanthranilic acid dioxygenase (HAAO) increases lifespan by 20-30% in C elegans. However, the mechanism of how these interventions may modulate response against different stressors during the aging process has yet to be explored. Fluorescent reporter strains show the stress-responsive transcription factors skn-1 (ortholog of NRF2/NFE2L2; oxidative stress response) and hif-1 (ortholog of HIF1A; hypoxic stress response) to be highly upregulated when the kynurenine pathway is inhibited. We also demonstrated the increase expression of gst-4 and gcs-1 (transcriptional targets skn-1), which are involved in production of the antioxidant glutathione (GSH), as well as upregulation of cysl-2 (transcriptional target of hif-1), a regulator of cysteine biosynthesis from serine. We hypothesize that lifespan extension resulting from kynurenine pathway inhibition is mediated, at least in part, by upregulation of these transcription factors, providing elevated defense against oxidative stress and hypoxic stress.

Low-Spin Cyanide Complexes of 3-Mercaptopropionic Acid Dioxygenase (MDO) Reveal the Impact of Outer-Sphere SHY-Motif Residues.

3-Mercaptopropionic acid (3MPA) dioxygenase (MDO) is a non-heme Fe(II)/O2-dependent oxygenase that catalyzes the oxidation of thiol-substrates to yield the corresponding sulfinic acid. Hydrogen-bonding interactions between the Fe-site and a conserved set of three outer-sphere residues (Ser-His-Tyr) play an important catalytic role in the mechanism of this enzyme. Collectively referred to as the SHY-motif, the functional role of these residues remains poorly understood. Here, catalytically inactive Fe(III)-MDO precomplexed with 3MPA was titrated with cyanide to yield a low-spin (S = 1/2) (3MPA/CN)-bound ternary complex (referred to as 1C). UV-visible and electron paramagnetic resonance (EPR) spectroscopy were used to monitor the binding of 3MPA and cyanide. Comparisons of results obtained from SHY-motif variants (H157N and Y159F) were performed to investigate specific H-bonding interactions. For the wild-type enzyme, the binding of 3MPA- and cyanide to the enzymatic Fe-site is selective and results in a homogeneous ternary complex. However, this selectivity is lost for the Y159F variant, suggesting that H-bonding interactions contributed from Tyr159 gate ligand coordination at the Fe-site. Significantly, the g-values for the low-spin ferric site are diagnostic of the directionality of Tyr159 H-bond donation. Computational models coupled with CASSCF/NEVPT2-calculated g-values were used to verify that a major shift in the central g-value (g2) displayed between wild-type and SHY variants could be attributed to the loss of Tyr159 H-bond donation to the Fe-bound cyanide. Applied to native cosubstrate, this H-bond donation provides a means to stabilize Fe-bound dioxygen and potentially explains the attenuated (∼15-fold) rate of catalytic turnover previously reported for MDO SHY-motif variants.

Rapid evaluation of the substrate specificity of 3-nitrobenzoic acid dioxygenase MnbAB via colorimetric detection using Saltzman reagent.

Nitroaromatic compounds are essential materials for chemical industry, but they are also potentially toxic environmental pollutants. Therefore, their sensitive detection and degradation are important concerns. The microbial degradation pathways of nitroaromatic compounds have been studied in detail, but their usefulness needs to be evaluated to understand their potential applications in bioremediation. Here, we developed a rapid and relatively sensitive assay system to evaluate the activities and substrate specificities of nitroaromatic dioxygenases involved in the oxidative biodegradation of nitroaromatic compounds. In this system, nitrous acid, which was released from the nitroaromatic compounds by the dioxygenases, was detected and quantified using the Saltzman reagent. Escherichia coli producing the 3-nitrobenzoic acid dioxygenase complex MnbAB from Comamonas sp. JS46 clearly showed the apparent substrate specificity of MnbAB as follows. MnbAB accepted not only 3-nitrobenzoic acid but also several other p- and m-nitrobenzoic acid derivatives as substrates, although it much preferred 3-nitrobenzoic acid to others. Furthermore, the presence of a hydroxy or an amino group at the ortho position of the nitro group decreased the activity of MnbAB. In addition, MnbAB accepted 2-(4-nitrophenyl)acetic acid as a substrate, which has one additional methylene group between the aromatic ring and the carboxy group of 3-nitrobenzoic acid. This is the first report about the detailed substrate specificity of MnbAB. Our system can be used for other nitroaromatic dioxygenases and contribute to their characterization.

Effects of α7 nicotinic receptor positive allosteric modulator PNU120596 on microglial quinolinic acid formation following LPS‐induced depressive‐like behavior in mice

Positive allosteric modulation of α7 nicotinic acetylcholine receptor (α7 nAChR) plays a critical role in regulating the neuroinflammation and microglial activation associated with major depressive disorder (MDD). Microglial quinolinic acid (QUIN), an N-methyl-D-aspartate (NMDA) receptor agonist, which is synthesized by 3-hydroxyanthranilic acid dioxygenase (HAAO) plays a critical role in the development of MDD-related symptoms. However, the role of α7 nAChR via allosteric modulation on QUIN formation in the brain in lipopolysaccharide (LPS)-induced depressive-like behavior is not known. Here, we have determined the effects of PNU120596, an α7 nAChR positive allosteric modulator, on HAAO expression and QUIN formation in the hippocampus and prefrontal cortex. We also examined the pharmacological interaction between PNU120596 and memantine, an NMDA receptor antagonist, on LPS-induced cognitive deficit and depressive-like behaviors. Immunofluorescence assay and real-time polymerase chain reaction were used to determine bionomical alteration, whereas Y-maze, tail suspension test (TST), and forced swim test (FST) were used to evaluate behavioral changes. LPS administration (1 mg/kg, i.p.) resulted in increased HAAO expression and QUIN formation in the hippocampus and prefrontal cortex. Pretreatment of PNU120596 (4 mg/kg, i.p.) reduced LPS-induced increase in HAAO expression and QUIN formation in these brain regions. Subthreshold dose of memantine (1 mg/kg, i.p.) enhanced the PNU120596 effects against LPS-induced cognitive deficit and depressive-like behaviors. Taken together, these results indicate that the antidepressant-like effects of PNU120596 are mediated by attenuation of LPS-induced QUIN formation in these brain regions. Therefore, α7 nAChR PAM could be a potential therapeutic utility for MDD associated with neurotoxic glutamatergic transmission.

A Yarrowia lipolytica Strain Engineered for Pyomelanin Production.

The yeast Yarrowia lipolytica naturally produces pyomelanin. This pigment accumulates in the extracellular environment following the autoxidation and polymerization of homogentisic acid, a metabolite derived from aromatic amino acids. In this study, we used a chassis strain optimized to produce aromatic amino acids for the de novo overproduction of pyomelanin. The gene 4HPPD, which encodes an enzyme involved in homogentisic acid synthesis (4-hydroxyphenylpyruvic acid dioxygenase), was characterized and overexpressed in the chassis strain with up to three copies, leading to pyomelanin yields of 4.5 g/L. Homogentisic acid is derived from tyrosine. When engineered strains were grown in a phenylalanine-supplemented medium, pyomelanin production increased, revealing that the yeast could convert phenylalanine to tyrosine, or that the homogentisic acid pathway is strongly induced by phenylalanine.

Genetically blocking HPD via CRISPR-Cas9 protects against lethal liver injury in a pig model of tyrosinemia type I

Hereditary tyrosinemia type I (HT1) results from the loss of fumarylacetoacetate hydrolase (FAH) activity and can lead to lethal liver injury (LLI). Therapeutic options for HT1 remain limited. The FAH−/− pig, a well-characterized animal model of HT1, represents a promising candidate for testing novel therapeutic approaches to treat this condition. Here, we report an improved single-step method to establish a biallelic (FAH−/−) mutant porcine model using CRISPR-Cas9 and cytoplasmic microinjection. We also tested the feasibility of rescuing HT1 pigs through inactivating the 4-hydroxyphenylpyruvic acid dioxygenase (HPD) gene, which functions upstream of the pathogenic pathway, rather than by directly correcting the disease-causing gene as occurs with traditional gene therapy. Direct intracytoplasmic delivery of CRISPR-Cas9 targeting HPD before intrauterine death reprogrammed the tyrosine metabolism pathway and protected pigs against FAH deficiency-induced LLI. Characterization of the F1 generation revealed consistent liver-protective features that were germline transmissible. Furthermore, HPD ablation ameliorated oxidative stress and inflammatory responses and restored the gene profile relating to liver metabolism homeostasis. Collectively, this study not only provided a novel large animal model for exploring the pathogenesis of HT1, but also demonstrated that CRISPR-Cas9-mediated HPD ablation alleviated LLI in HT1 pigs and represents a potential therapeutic option for the treatment of HT1.

Structure of 3-mercaptopropionic acid dioxygenase with a substrate analog reveals bidentate substrate binding at the iron center

Thiol dioxygenases are a subset of nonheme iron oxygenases that catalyze the formation of sulfinic acids from sulfhydryl-containing substrates and dioxygen. Among this class, cysteine dioxygenases (CDOs) and 3-mercaptopropionic acid dioxygenases (3MDOs) are the best characterized, and the mode of substrate binding for CDOs is well understood. However, the manner in which 3-mercaptopropionic acid (3MPA) coordinates to the nonheme iron site in 3MDO remains a matter of debate. A model for bidentate 3MPA coordination at the 3MDO Fe-site has been proposed on the basis of computational docking, whereas steady-state kinetics and EPR spectroscopic measurements suggest a thiolate-only coordination of the substrate. To address this gap in knowledge, we determined the structure of Azobacter vinelandii 3MDO (Av3MDO) in complex with the substrate analog and competitive inhibitor, 3-hydroxypropionic acid (3HPA). The structure together with DFT computational modeling demonstrates that 3HPA and 3MPA associate with iron as chelate complexes with the substrate-carboxylate group forming an additional interaction with Arg168 and the thiol bound at the same position as in CDO. A chloride ligand was bound to iron in the coordination site assigned as the O2-binding site. Supporting HYSCORE spectroscopic experiments were performed on the (3MPA/NO)-bound Av3MDO iron nitrosyl (S = 3/2) site. In combination with spectroscopic simulations and optimized DFT models, this work provides an experimentally verified model of the Av3MDO enzyme–substrate complex, effectively resolving a debate in the literature regarding the preferred substrate-binding denticity. These results elegantly explain the observed 3MDO substrate specificity, but leave unanswered questions regarding the mechanism of substrate-gated reactivity with dioxygen.

Coexpression Clusters and Allele-Specific Expression in Metabolism-Based Herbicide Resistance.

In the last decade, Amaranthus tuberculatus has evolved resistance to 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-hydroxyphenylpyruvate dioxygenase inhibitors in multiple states across the midwestern United States. Two populations resistant to both mode-of-action groups, one from Nebraska (NEB) and one from Illinois (CHR), were studied using an RNA-seq approach on F2 mapping populations to identify the genes responsible for resistance. Using both an A. tuberculatus transcriptome assembly and a high-quality grain amaranth (A. hypochondriacus) genome as references, differential transcript and gene expression analyses were conducted to identify genes that were significantly over- or underexpressed in resistant plants. When these differentially expressed genes (DEGs) were mapped on the A. hypochondriacus genome, physical clustering of the DEGs was apparent along several of the 16 A. hypochondriacus scaffolds. Furthermore, single-nucleotide polymorphism calling to look for resistant-specific (R) variants, and subsequent mapping of these variants, also found similar patterns of clustering. Specifically, regions biased toward R alleles overlapped with the DEG clusters. Within one of these clusters, allele-specific expression of cytochrome P450 81E8 was observed for 2,4-D resistance in both the CHR and NEB populations, and phylogenetic analysis indicated a common evolutionary origin of this R allele in the two populations.

Co-treatments to Boost IDO Activity and Inhibit Production of Downstream Catabolites Induce Durable Suppression of Experimental Autoimmune Encephalomyelitis.

Reinforcing defective tolerogenic processes slows progression of autoimmune (AI) diseases and has potential to promote drug-free disease remission. Previously, we reported that DNA nanoparticles (DNPs) and cyclic dinucleotides (CDNs) slow progression of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, by activating the Stimulator of Interferon Genes (STING) signaling adaptor to stimulate interferon type 1 (IFN-I) production, which induced dendritic cells to express indoleamine 2,3 dioxygenase (IDO) and acquire immune regulatory phenotypes. Here, we show that therapeutic responses to DNPs depend on DNA sensing via cyclic GAMP synthase (cGAS) and interactions between Programmed Death-1 (PD-1) and PD-1 ligands. To investigate how increased tryptophan (Trp) metabolism by IDO promotes therapeutic responses mice were co-treated at EAE onset with DNPs and drugs that inhibit kynurenine aminotransferase-II (KatII) or 3-hydroxyanthranilic acid dioxygenase (HAAO) activity downstream of IDO in the kynurenine (Kyn) pathway. DNP and KatII or HAAO inhibitor co-treatments suppressed EAE progression more effectively than DNPs, while KatII inhibition had no significant therapeutic benefit and HAAO inhibition attenuated but did not prevent EAE progression. Moreover, therapeutic responses to co-treatments were durable as EAE progression did not resume after co-treatment. Thus, using STING agonists to boost IDO activity and manipulating the Kyn pathway downstream of IDO is an effective strategy to enhance tolerogenic responses that overcome autoimmunity to suppress EAE progression.

Paraburkholderia madseniana sp. nov., a phenolic acid-degrading bacterium isolated from acidic forest soil.

RP11T was isolated from forest soil following enrichment with 4-hydroxybenzoic acid. Cells of RP11T are aerobic, non-sporulating, exhibit swimming motility, and are rods (0.8 µm by 1.4 µm) that often occur as diplobacillus or in short chains (3-4 cells). Optimal growth on minimal media containing 4-hydroxybenzoic acid (µ=0.216 hr-1) occurred at 30 °C, pH 6.5 or 7.0 and 0% salinity. Comparative chemotaxonomic, genomic and phylogenetic analyses revealed the isolate was distinct from its closest relative type strains identified as Paraburkholderia aspalathi LMG 27731T, Paraburkholderia fungorum LMG 16225T and Paraburkholderia caffeinilytica CF1T. Strain RP11T is genetically distinct from P. aspalathi, its closest relative, in terms of 16S rRNA gene sequence similarity (98.7%), genomic average nucleotide identity (94%) and in silico DNA-DNA hybridization (56.7 %±2.8). The composition of fatty acids and substrate utilization pattern differentiated strain RP11T from its closest relatives, including growth on phthalic acid. Strain RP11T encoded the greatest number of aromatic degradation genes of all eleven closely related type strains and uniquely encoded a phthalic acid dioxygenase and paralog of the 3-hydroxybenzoate 4-monooxygenase. The only ubiquinone detected in strain RP11T was Q-8, and the major cellular fatty acids were C16 : 0, 3OH-C16 : 0, C17 : 0 cyclo, C19 : 0 cyclo ω8c, and summed feature 8 (C18 : 1 ω7c/ω6c). On the basis of this polyphasic approach, it was determined that strain RP11T represents a novel species from the genus Paraburkholderia for which the name Paraburkholderia madseniana sp. nov. is proposed. The type strain is RP11T (=DSM 110123T=LMG 31517T).