Acid Hydroxylase Research Articles

Aromatic Amino Acid Hydroxylases as Off-Targets of Histone Deacetylase Inhibitors

Aromatic Amino Acid Hydroxylases as Off-Targets of Histone Deacetylase Inhibitors

The Synergistic and Opposing Roles of ω-Fatty Acid Hydroxylase (CYP4A11) and ω-1 Fatty Acid Hydroxylase (CYP2E1) in Chronic Liver Disease

Cytochrome P450 fatty acid hydroxylase consists of members of the CYP4 family that ω-hydroxylate fatty acids and the CYP2E1 that ω-1 hydroxylates fatty acids. Although ω and ω-1 hydroxylation of fatty acids have been thought to play a minor role in fatty acid metabolism (less than 20%), it plays a vital role in excess liver fatty acids overload seen in fasting, diabetes, metabolic disorder, and over-consumption of alcohol and high-fat diet. This pathway provides anabolic metabolites for gluconeogenesis, succinate, and acetate for lipogenesis. The CYP4A and CYP2E1 genes are activated in fasting and several metabolic disorders, suggesting a synergistic role in preventing fatty acid-induced lipotoxicity with the consequence of increased liver cholesterol and lipogenesis leading to increased Lipid Droplet (LD) deposition. During the progression of Metabolic Dysfunction-associated Steatotic Liver Disease (MASLD), activation of Phospholipase A2 (PLA2) releases arachidonic acid that CYP4A11 and CYP2E1 P450s metabolize to produce 20-hydroxyeicosatetraenoic acid (20-HETE) and 19-HETE, respectively. These metabolites have opposing roles in the progression of MASLD and chronic liver disease (CLD). This report discusses the synergistic role of the CYP4A and CYP2E1 P450s in the metabolism of saturated and unsaturated fatty acids and their opposite physiological role in the metabolism of Arachidonic Acid (AA). We finally discuss the role of ethanol in disrupting the synergistic and opposing roles of the CYP4A and CYP2E1 genes in MASLD and CLD.

Hydroxy Fatty Acid Synthesis-Related mRNA as the Biomarker for Detecting Mislabeling of Honey Entomological Origin.

The authentication of the entomological origin of honey is a widespread concern, necessitating the prompt establishment of an effective approach for distinguishing between Apis cerana cerana honey (ACH) and Apis mellifera ligustica honey (AMH). Hydroxy fatty acids (HFAs) found in honey are bee-derived components synthesized by the mandibular glands of worker bees. We previously discovered significant variations in the hydroxy fatty acid composition between ACH and AMH, suggesting their potential as indicators for identifying the authenticity of the entomological origin of honey. Herein, we identified differentially expressed genes associated with HFA synthesis by conducting transcriptome sequencing of the mandibular glands of AC and AM honeybees. Subsequently, we proposed a method for the relative quantitative analysis of bee-derived RNA components using real-time fluorescence quantitative polymerase chain reaction, which was supplemented by multivariate statistical analysis to further discern differences in HFA synthesis-related mRNA between ACH and AMH. The results showed that the mRNAs of FAXDC2 (fatty acid hydroxylase domain-containing protein 2) and FAS (fatty acid synthase) may serve as indicators to discern the entomological origin of honey. This study presents two novel biomarkers for detecting mislabeling of the entomological origin in ACH and AMH based on variations in bee-derived components.

Characterization of Dnajc12 knockout mice, a model of hypodopaminergia.

Homozygous DNAJC12 c.79-2A>G (p. V27Wfs*14) loss-of-function mutations were first reported as a cause of young-onset Parkinson's disease. However, bi-allelic autosomal recessive pathogenic variants in DNAJC12 may lead to an alternative constellation of neurological features including infantile dystonia, developmental delay, intellectual disability and neuropsychiatric disorders. DNAJC12 is understood to co-chaperone aromatic amino acid hydroxylases to enhance the synthesis of biogenic amines. In vitro , we confirm overexpressed DNAJC12 forms a complex with tyrosine hydroxylase, the rate-limiting enzyme in dopamine (DA) synthesis. Now we describe a conditional knockout mouse (cDKO) in which loxP sites flanking Dnajc12 exon 2 enable its excision by cre-recombinase to create a constitutive Dnajc12 knock out (DKO). At three months of age, DKO animals exhibit reduced locomotion and exploratory behavior in automated open-field testing. DKO mice also manifest increased plasma phenylalanine levels, a cardinal feature of patients with DNAJC12 pathogenic variants. In striatal tissue, total DA and serotonin, and their metabolites, are reduced. Biochemical alterations in synaptic proteins and tyrosine hydroxylase are also apparent, with enhanced phosphorylation of pSer31 and pSer40 sites that may reflect biological compensation. Electrically-evoked striatal DA is reduced. Most immediately, cDKO and DKO mice present models to develop and refined therapeutic approaches for the treatment of DNAJC12 dystonia and parkinsonism. These models may also enable the pleiotropic functions of biogenic amines (including DA) to be individually investigated in the brain and periphery.

Prolonged xenokidney graft survival in sensitized NHP recipients by expression of multiple human transgenes in a triple knockout pig.

Genetic modification of porcine donors, combined with optimized immunosuppression, has been shown to improve outcomes of experimental xenotransplant. However, little is known about outcomes in sensitized recipients, a population that could potentially benefit the most from the clinical implementation of xenotransplantation. Here, five highly allosensitized rhesus macaques received a porcine kidney from GGTA1 (α1,3-galactosyltransferase) knockout pigs expressing the human CD55 transgene (1KO.1TG) and were maintained on an anti-CD154 monoclonal antibody (mAb)-based immunosuppressive regimen. These recipients developed de novo xenoreactive antibodies and experienced xenograft rejection with evidence of thrombotic microangiopathy and antibody-mediated rejection (AMR). In comparison, three highly allosensitized rhesus macaques receiving a kidney from GGTA1, CMAH (cytidine monophospho-N-acetylneuraminic acid hydroxylase), and b4GNT2/b4GALNT2 (β-1,4-N-acetyl-galactosaminyltransferase 2) knockout pigs expressing seven human transgenes including human CD46, CD55, CD47, THBD (thrombomodulin), PROCR (protein C receptor), TNFAIP3 (tumor necrosis factor-α-induced protein 3), and HMOX1 (heme oxygenase 1) (3KO.7TG) experienced significantly prolonged graft survival and reduced AMR, associated with dampened post-transplant humoral responses, early monocyte and neutrophil activation, and T cell repopulation. After withdrawal of all immunosuppression, recipients who received kidneys from 3KO.7TG pigs rejected the xenografts via AMR. These data suggest that allosensitized recipients may be suitable candidates for xenografts from genetically modified porcine donors and could benefit from an optimized immunosuppression regimen designed to target the post-transplant humoral response, thereby avoiding AMR.

Anatomical organization of the cerebrum of the praying mantis Hierodula membranacea.

Many predatory animals, such as the praying mantis, use vision for prey detection and capture. Mantises are known in particular for their capability to estimate distances to prey by stereoscopic vision. While the initial visual processing centers have been extensively documented, we lack knowledge on the architecture of central brain regions, pivotal for sensory motor transformation and higher brain functions. To close this gap, we provide a three-dimensional (3D) reconstruction of the central brain of the Asian mantis, Hierodula membranacea. The atlas facilitates in-depth analysis of neuron ramification regions and aides in elucidating potential neuronal pathways. We integrated seven 3D-reconstructed visual interneurons into the atlas. In total, 42 distinct neuropils of the cerebrum were reconstructed based on synapsin-immunolabeled whole-mount brains. Backfills from the antenna and maxillary palps, as well as immunolabeling of γ-aminobutyric acid (GABA) and tyrosine hydroxylase (TH), further substantiate the identification and boundaries of brain areas. The composition and internal organization of the neuropils were compared to the anatomical organization of the brain of the fruit fly (Drosophila melanogaster) and the two available brain atlases of Polyneoptera-the desert locust (Schistocerca gregaria) and the Madeira cockroach (Rhyparobia maderae). This study paves the way for detailed analyses of neuronal circuitry and promotes cross-species brain comparisons. We discuss differences in brain organization between holometabolous and polyneopteran insects. Identification of ramification sites of the visual neurons integrated into the atlas supports previous claims about homologous structures in the optic lobes of flies and mantises.

Draft genome sequence of potentially dikaryotic black fungus Aureobasidium melanogenum isolated from aircraft.

The Ascomycota yeast Aureobasidium melanogenum strain W12 was isolated from an aircraft polymer-coated surface. The genome size is 53,160,883 bp with a G + C content of 50.13%. The genome contains fatty acid transporters, cutinases, hydroxylases, and lipases potentially used for survival on polymer coatings on aircraft.

The cholesterol biosynthesis enzyme FAXDC2 couples Wnt/β-catenin to RTK/MAPK signaling.

Wnts, cholesterol, and MAPK signaling are essential for development and adult homeostasis. Here, we report that fatty acid hydroxylase domain containing 2 (FAXDC2), a previously uncharacterized enzyme, functions as a methyl sterol oxidase catalyzing C4 demethylation in the Kandutsch-Russell branch of the cholesterol biosynthesis pathway. FAXDC2, a paralog of MSMO1, regulated the abundance of the specific C4-methyl sterols lophenol and dihydro-T-MAS. Highlighting its clinical relevance, FAXDC2 was repressed in Wnt/β-catenin-high cancer xenografts, in a mouse genetic model of Wnt activation, and in human colorectal cancers. Moreover, in primary human colorectal cancers, the sterol lophenol, regulated by FAXDC2, accumulated in the cancerous tissues and not in adjacent normal tissues. FAXDC2 linked Wnts to RTK/MAPK signaling. Wnt inhibition drove increased recycling of RTKs and activation of the MAPK pathway, and this required FAXDC2. Blocking Wnt signaling in Wnt-high cancers caused both differentiation and senescence; and this was prevented by knockout of FAXDC2. Our data show the integration of 3 ancient pathways, Wnts, cholesterol synthesis, and RTK/MAPK signaling, in cellular proliferation and differentiation.

Mouse models for inherited monoamine neurotransmitter disorders.

Several mouse models have been developed to study human defects of primary and secondary inherited monoamine neurotransmitter disorders (iMND). As the field continues to expand, current defects in corresponding mouse models include enzymes and a molecular co-chaperone involved in monoamine synthesis and metabolism (PAH, TH, PITX3, AADC, DBH, MAOA, DNAJC6), tetrahydrobiopterin (BH4) cofactor synthesis and recycling (adGTPCH1/DRD, arGTPCH1, PTPS, SR, DHPR), and vitamin B6 cofactor deficiency (ALDH7A1), as well as defective monoamine neurotransmitter packaging (VMAT1, VMAT2) and reuptake (DAT). No mouse models are available for human DNAJC12 co-chaperone and PNPO-B6 deficiencies, disorders associated with recessive variants that result in decreased stability and function of the aromatic amino acid hydroxylases and decreased neurotransmitter synthesis, respectively. More than one mutant mouse is available for some of these defects, which is invaluable as different variant-specific (knock-in) models may provide more insights into underlying mechanisms of disorders, while complete gene inactivation (knock-out) models often have limitations in terms of recapitulating complex human diseases. While these mouse models have common phenotypic traits also observed in patients, reflecting the defective homeostasis of the monoamine neurotransmitter pathways, they also present with disease-specific manifestations with toxic accumulation or deficiency of specific metabolites related to the specific gene affected. This review provides an overview of the currently available models and may give directions toward selecting existing models or generating new ones to investigate novel pathogenic mechanisms and precision therapies.

Tuning the peroxidase activity of artificial P450 peroxygenase by engineering redox-sensitive residues.

Cytochrome P450 monooxygenases (P450s) are well recognized as versatile bio-oxidation catalysts. However, the catalytic functions of P450s are highly dependent on NAD(P)H and redox partner proteins. Our group has recently reported the use of a dual-functional small molecule (DFSM) for generating peroxygenase activity of P450BM3, a long-chain fatty acid hydroxylase from Bacillus megaterium. The DFSM-facilitated P450BM3 peroxygenase system exhibited excellent peroxygenation activity and regio-/enantioselectivity for various organic substrates, such as styrenes, thioanisole, small alkanes, and alkylbenzenes. Very recently, we demonstrated that the DFSM-facilitated P450BM3 peroxygenase could be switched to a peroxidase by engineering the redox-sensitive tyrosine residues in P450BM3. Given the great potential of P450 peroxidase for C-H oxyfunctionalization, we herein report scrutiny of the effect of mutating redox-sensitive residues on peroxidase activity by deeply screening all redox-sensitive residues of P450BM3, namely methionines, tryptophans, cysteines, and phenylalanines. As a result, six beneficial mutations at positions M212, F81, M112, F173, M177, and F77 were screened out from 78 constructed mutants, and significantly enhanced the peroxidase activity of P450BM3 in the presence of Im-C6-Phe, a typical DFSM molecule. Further combination of the beneficial mutations resulted in a more than 100-fold improvement in peroxidase activity compared with that of the combined parent enzyme and DFSM, comparable to or better than most natural peroxidases. In addition, mutations of redox-sensitive residues even dramatically increased, by more than 300-fold, the peroxidase activity of the starting F87A enzyme in the absence of the DFSM, despite the far lower apparent catalytic turnover number compared with the DFSM-P450 system. This study provides new insights and a potential strategy for regulating the catalytic promiscuity of P450 enzymes for multiple functional oxidations.

Thermostable fatty acid hydroxylases from ancestral reconstruction of cytochrome P450 family 4 enzymes

Ancestral sequence reconstruction can be used to obtain highly thermostable catalysts of fatty acid hydroxylation.

LC-MS/MS methods for direct measurement of sepiapterin and tetrahydrobiopterin in human plasma and clinical applications.

Aim: Tetrahydrobiopterin (BH4), a natural cofactor of aromatic amino acid hydroxylases, and sepiapterin, a natural precursor of BH4, are endogenously present in human plasma. This is the first report on methods for direct quantification of sepiapterin and BH4 in human plasma by LC-MS/MS for pharmacokinetic assessment. Materials & methods: The analytes in plasma were harvested from blood that were treated with 10% ascorbic acid (AA) to a final concentration of 1% AA. Results & conclusion: The quantification methods were validated for calibration ranges of 0.75-500ng/ml and 0.5-500ng/ml for sepiapterin and BH4, respectively. Quantification of analytes was challenging due to their susceptibility to redox reactions. The validated methods were utilized successfully to support clinical development of sepiapterin.

Postharvest caffeic acid dipping enhances disease resistance and storage capacity of ‘Zaosu’ pear fruit via regulating phenylpropane metabolism

Caffeic acid, a naturally occurring and safe phenolic compound, acts as a precursor in lignin synthesis within the phenylpropane metabolism pathway. The aims of this study was to evaluate the impacts of postharvest caffeic acid immersion on Alternaria rot and postharvest quality of ‘Zaosu’ pear fruit, as well as elucidate the underlying mechanisms involved. Results showed that postharvest caffeic acid dipping effectively inhibited spore germination and colony diameter of Alternaria alternata, as well as lesion diameter in A. alternata-inoculated pears. Caffeic acid treatment also delayed the increase of weight loss and decline in flesh firmness, increased total soluble solids content, and reduced surface yellowing of pears. Additionally, caffeic acid treatment enhanced the enzymatic activities and gene transcription levels of phenylalanine ammonialyase, 4-coumarate/coenzyme A ligase, cinnamate 4-hydroxylase, peroxidase, cinnamyl alcohol dehydrogenase, and polyphenol oxidase in pears. Meanwhile, the transcription levels of p-coumarate-3-hydroxylase, caffeic acid O-methyltransferase, cinnamoyl CoA reductase, caffeoyl shikimate esterase, hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase, caffeoyl CoA O-methyltransferase, ferulic acid 5-hydroxylase, and laccase genes were also up-regulated by caffeic acid treatment in pears. Furthermore, caffeic acid dipping enhanced phenylalanine, flavonoids, caffeic acid, total phenolic compounds, and lignin levels in the fruit. Therefore, caffeic acid exhibits promising potential for enhancing resistance against A. alternata and maintaining the storage capacity of pears via inducing phenylpropane metabolism.

FAXDC2 inhibits the proliferation and invasion of human liver cancer HepG2cells.

The reprogramming of lipid metabolism serves an important role in occurrence and development of liver cancer. Fatty acid hydroxylase domain containing 2 (FAXDC2) is a hydroxylase involved in the synthesis of cholesterol and sphingomyelin and downregulated in various types of cancer. There are no reports on the relationship between FAXDC2 and liver carcinogenesis. The present study used multiple portals and publicly available tools to explore its correlation with liver cancer. The results showed that the expression of FAXDC2 decreased in liver cancer and the methylation level near the promoter increased. Patients with liver cancer and with low expression of FAXDC2 had a poor prognosis. Gain of function and loss of function strategies were performed to evaluate its roles in liver cancer cells. CCK-8 assay showed that overexpression of FAXDC2 inhibited the viability of liver cancer cells (HepG2). Flow cytometry analysis indicated that HepG2 cells with overexpressing FAXDC2 showed an S phase arrest, associated with cyclin-dependent kinase 2 decreased. Transwell experiments showed that increasing FAXDC2 inhibited HepG2 cell invasion ability, accompanied by the upregulation of E-cadherin. Notably, knockdown of FAXDC2 had no significant effect on cell cycle and invasion functions. Based on the cBioPortal platform, FAXDC2 was predicted to closely correlate to the ERK signal in tumorigenesis. Western blotting results showed that overexpression of FAXDC2 decreased the phosphorylation level of ERK in liver cancer cells. The present study first identified FAXDC2 as a liver cancer suppressor, which might inhibit the proliferation and invasion of liver cancer cells through the mechanism associated with ERK signaling. The present study provided a possible new target for the diagnosis and treatment of liver cancer.

Cmah deficiency blunts cellular senescence in adipose tissues and improves whole-body glucose metabolism in aged mice.

Cytidine monophosphate-N-acetylneuraminic acid (Neu5Ac) hydroxylase (Cmah) is an enzyme, which converts Neu5Ac to the sialic acid Neu5Gc. Neu5Gc is thought to increase inflammatory cytokines, which are, in part, produced in senescent cells of adipose tissues. Cellular senescence in adipose tissues induces whole-body aging and impaired glucose metabolism. Therefore, we hypothesized that Cmah deficiency would prevent cellular senescence in adipose tissues and impaired glucose metabolism. Wild-type (WT) and Cmah knockout (KO) mice aged 24-25 months were used. Whole-body metabolism was assessed using a metabolic gas analysis system. We measured blood glucose and insulin concentrations after oral glucose administration. The size of the lipid droplets in the liver was quantified. Markers of cellular senescence and senescence-associated secretory phenotypes were measured in adipose tissues. Cmah KO had significantly increased VO2 and energy expenditure (P < 0.01). Unlike glucose, the insulin concentration after oral glucose administration was significantly lower in the Cmah KO group than in the WT group (P < 0.001). Lipid droplets in the liver were significantly lower in the Cmah KO group than in the WT group (P < 0.05). The markers of cellular senescence and senescence-associated secretory phenotypes in the adipose tissues were significantly lower in the Cmah KO group than in the WT group (P < 0.05). Cmah deficiency blunted cellular senescence in adipose tissues and improved whole-body glucose metabolism. These characteristics in aged Cmah KO mice might be associated with higher energy expenditure. Geriatr Gerontol Int 2023; 23: 958-964.

In Vitro and In Vivo Chaperone Effect of (R)-2-amino-6-(1R, 2S)-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin-4(3H)-one on the C1473G Mutant Tryptophan Hydroxylase 2.

Tryptophan hydroxylase 2 (TPH2) is the key and rate-limiting enzyme of serotonin (5-HT) synthesis in the mammalian brain. The 1473G mutation in the Tph2 gene decreases TPH2 activity in the mouse brain by twofold. (R)-2-amino-6-(1R, 2S)-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin-4(3H)-one (BH4) is a pharmacological chaperone for aromatic amino acid hydroxylases. In the present study, chaperone effects of BH4 on the mutant C1473G TPH2 were investigated in vitro and in vivo. In vitro BH4 increased the thermal stability (T50 value) of mutant and wild-type TPH2 molecules. At the same time, neither chronic (twice per day for 7 days) intraperitoneal injection of 48.3 mg/kg of BH4 nor a single intraventricular administration of 60 μg of the drug altered the mutant TPH2 activity in the brain of Balb/c mice. This result indicates that although BH4 shows a chaperone effect in vitro, it is unable to increase the activity of mutant TPH2 in vivo.

Catalytic Site Constraints in the P450s Mediating Loganic Acid (7DLH) and Secologanic Acid Synthesis (SLAS) in Camptotheca.

Terpene indole alkaloids (TIAs) are plant-derived natural products synthesized in low levels in medicinal plants such as Catharanthus roseus and Camptotheca acuminata. TIA pathways species utilize several CYP72A subfamily members to form loganic acid from 7-deoxyloganic acid (a simple hydroxylation) as well as secologanin and secologanic acid from loganin and loganic acid (a C-C bond scission). Divergences in the specificities of these P450s have allowed Camptotheca secologanic acid synthases (SLASs) to become bifunctional enzymes capable of performing both reactions. In contrast, Catharanthus 7-deoxyloganic acid hydroxylase (7DLH) and secologanin synthase (SLS) have remained monofunctional enzymes capable either of monooxygenation or C-C bond scission. Our in vitro reconstitutions have now demonstrated that Camptotheca also contains a monofunctional 7DLH capable only of hydroxylating 7-deoxyloganic acid. Mutageneses aimed at evaluating residues important for the tight specificity of Camptotheca 7DLH (CYP72A729) and the broad specificity of SLAS (CYP72A564) have identified several residues where reciprocal switches substantially affect their activities: Lys128His in 7DLH increases hydroxylation of 7-deoxyloganic acid, and His132Lys in SLAS decreases this hydroxylation and C-C bond scissions of loganic acid and loganin; Gly321Ser in 7DLH does not affect hydroxylation of 7-deoxyloganic acid, whereas Ser324Gly in SLAS significantly increases C-C bond scission of loganic acid; Asp332Glu in the acid-alcohol pair of 7DLH increases hydroxylation of 7-deoxyloganic acid, whereas Glu335Asp in SLAS completely eliminates both of its activities. These mutations that enhance or eliminate these respective activities have significant potential to aid engineering efforts aimed at increasing TIA production in cell cultures, microbial systems, and/or other plants.

Activated Expression of Rice DMR6-like Gene OsS3H Partially Explores the Susceptibility to Bacterial Leaf Streak Mediated by Knock-Out OsF3H04g.

Downy Mildew Resistance 6-like (DMR6-like) genes are identified as salicylic acid (SA) hydroxylases and negative regulators of plant immunity. Previously, we identified two rice DMR6-like genes, OsF3H03g, and OsF3H04g, that act as susceptible targets of transcription activator-like effectors (TALEs) from Xanthomonas oryzae pv. oryzicola (Xoc), which causes bacterial leaf streak (BLS) in rice. Furthermore, all four homologs of rice DMR6-like proteins were identified to predominantly carry the enzyme activity of SA 5-hydroxylase (S5H), negatively regulate rice broad-spectrum resistance, and cause the loss of function of these OsDMR6s, leading to increased resistance to rice blast and bacterial blight (BB). Here, we curiously found that an OsF3H04g knock-out mutant created by T-DNA insertion, osf3h04g, was remarkedly susceptible to BLS and BB and showed an extreme reduction in SA content. OsF3H04g knock-out rice lines produced by gene-editing were mildly susceptible to BLS and reduced content of SA. To explore the susceptibility mechanism in OsF3H04g loss-of-function rice lines, transcriptome sequencing revealed that another homolog, OsS3H, had induced expression in the loss-of-function OsF3H04g rice lines. Furthermore, we confirmed that a great induction of OsS3H downstream and genomically adjacent to OsF3H04g in osf3h04g was primarily related to the inserted T-DNA carrying quadruple enhancer elements of 35S, while a slight induction was caused by an unknown mechanism in gene-editing lines. Then, we found that the overexpression of OsS3H increased rice susceptibility to BLS, while gene-editing mediated the loss-of-function OsS3H enhanced rice resistance to BLS. However, the knock-out of both OsF3H04g and OsS3H by gene-editing only neutralized rice resistance to BLS. Thus, we concluded that the knock-out of OsF3H04g activated the expression of the OsS3H, partially participating in the susceptibility to BLS in rice.

Enzymatic Hydroxylation of Aliphatic C-H Bonds by a Mn/Fe Cofactor.

The aerobic oxidation of carbon-hydrogen (C-H) bonds in biology is currently known to be accomplished by a limited set of cofactors that typically include heme, nonheme iron, and copper. While manganese cofactors perform difficult oxidation reactions, including water oxidation within Photosystem II, they are generally not known to be used for C-H bond activation, and those that do catalyze this important reaction display limited intrinsic reactivity. Here we report that the 2-aminoisobutyric acid hydroxylase from Rhodococcus wratislaviensis, AibH1H2, requires manganese to functionalize a strong, aliphatic C-H bond (BDE = 100 kcal/mol). Structural and spectroscopic studies of this enzyme reveal a redox-active, heterobimetallic manganese-iron active site at the locus of O2 activation and substrate coordination. This result expands the known reactivity of biological manganese-iron cofactors, which was previously restricted to single-electron transfer or stoichiometric protein oxidation. Furthermore, the AibH1H2 cofactor is supported by a protein fold distinct from typical bimetallic oxygenases, and bioinformatic analyses identify related proteins abundant in microorganisms. This suggests that many uncharacterized monooxygenases may similarly require manganese to perform oxidative biochemical tasks.

CRISPR/Cas9-mediated simultaneous mutation of threesalicylic acid 5-hydroxylase (OsS5H) genes confers broad-spectrum disease resistance in rice.

Salicylic acid (SA) is an essential plant hormone that plays critical roles in basal defence and amplification of local immune responses and establishes resistance against various pathogens. However, the comprehensive knowledge of the salicylic acid 5-hydroxylase (S5H) in rice-pathogen interaction is still elusive. Here, we reported that three OsS5H homologues displayed salicylic acid 5-hydroxylase activity, converting SA into 2,5-dihydroxybenzoic acid (2,5-DHBA). OsS5H1, OsS5H2, and OsS5H3 were preferentially expressed in rice leaves at heading stage and responded quickly to exogenous SA treatment. We found that bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) strongly induced the expression of OsS5H1, OsS5H2, and OsS5H3. Rice plants overexpressing OsS5H1, OsS5H2, and OsS5H3 showed significantly decreased SA contents and increased 2,5-DHBA levels, and were more susceptible to bacterial blight and rice blast. A simple single guide RNA (sgRNA) was designed to create oss5h1oss5h2oss5h3 triple mutants through CRISPR/Cas9-mediated gene mutagenesis. The oss5h1oss5h2oss5h3 exhibited stronger resistance to Xoo than single oss5h mutants. And oss5h1oss5h2oss5h3 plants displayedenhanced rice blast resistance. The conferred pathogen resistance in oss5h1oss5h2oss5h3 wasattributed to the significantly upregulation of OsWRKY45 and pathogenesis-related (PR) genes. Besides, flg22-induced reactive oxygen species (ROS) burst was enhanced in oss5h1oss5h2oss5h3. Collectively, our study provides a fast and effective approach to generate rice varieties with broad-spectrum disease resistance through OsS5H gene editing.