Biochemical and Structural Characterization of OvoATh2: A Mononuclear Nonheme Iron Enzyme from Hydrogenimonas thermophila for Ovothiol Biosynthesis.

Ovothiol A and ergothioneine are thiol-histidine derivatives with sulfur substitutions at the δ-carbon or ε-carbon of the l-histidine imidazole ring, respectively. Both ovothiol A and ergothioneine have protective effects on many aging-related diseases, and the sulfur substitution plays a key role in determining their chemical and biological properties, while factors governing sulfur incorporation regioselectivities in ovothiol and ergothioneine biosynthesis in the corresponding enzymes (OvoA, Egt1, or EgtB) are not yet known. In this study, we have successfully obtained the first OvoA crystal structure, which provides critical information to explain their C-S bond formation regioselectivity. Furthermore, OvoATh2 exhibits several additional activities: (1) ergothioneine sulfoxide synthase activity akin to Egt1 in ergothioneine biosynthesis; (2) cysteine dioxygenase activity using l-cysteine and l-histidine analogues as substrates; (3) cysteine dioxygenase activity upon mutation of an active site tyrosine residue (Y406). The structural insights and diverse chemistries demonstrated by OvoATh2 pave the way for future comprehensive structure-function correlation studies.

An S=1 Iron(IV) Intermediate Revealed in a Non-Heme Iron Enzyme-Catalyzed Oxidative C-S Bond Formation.

Ergothioneine (ESH) and ovothiol A (OSHA) are two natural thiol-histidine derivatives. ESH has been implicated as a longevity vitamin and OSHA inhibits the proliferation of hepatocarcinoma. The key biosynthetic step of ESH and OSHA in the aerobic pathways is the O2 -dependent C-S bond formation catalyzed by non-heme iron enzymes (e.g., OvoA in ovothiol biosynthesis), but due to the lack of identification of key reactive intermediate the mechanism of this novel reaction is unresolved. In this study, we report the identification and characterization of a kinetically competent S=1 iron(IV) intermediate supported by a four-histidine ligand environment (three from the protein residues and one from the substrate) in enabling C-S bond formation in OvoA from Methyloversatilis thermotoleran, which represents the first experimentally observed intermediate spin iron(IV) species in non-heme iron enzymes. Results reported in this study thus set the stage to further dissect the mechanism of enzymatic oxidative C-S bond formation in the OSHA biosynthesis pathway. They also afford new opportunities to study the structure-function relationship of high-valent iron intermediates supported by a histidine rich ligand environment.

Dissecting the Mechanism of the Nonheme Iron Endoperoxidase FtmOx1 Using Substrate Analogues.

FtmOx1 is a nonheme iron (NHFe) endoperoxidase, catalyzing three disparate reactions, endoperoxidation, alcohol dehydrogenation, and dealkylation, under in vitro conditions; the diversity complicates its mechanistic studies. In this study, we use two substrate analogues to simplify the FtmOx1-catalyzed reaction to either a dealkylation or an alcohol dehydrogenation reaction for structure-function relationship analysis to address two key FtmOx1 mechanistic questions: (1) Y224 flipping in the proposed COX-like model vs α-ketoglutarate (αKG) rotation proposed in the CarC-like mechanistic model and (2) the involvement of a Y224 radical (COX-like model) or a Y68 radical (CarC-like model) in FtmOx1-catalysis. When 13-oxo-fumitremorgin B (7) is used as the substrate, FtmOx1-catalysis changes from the endoperoxidation to a hydroxylation reaction and leads to dealkylation. In addition, consistent with the dealkylation side-reaction in the COX-like model prediction, the X-ray structure of the FtmOx1•CoII•αKG•7 ternary complex reveals a flip of Y224 to an alternative conformation relative to the FtmOx1•FeII•αKG binary complex. Verruculogen (2) was used as a second substrate analogue to study the alcohol dehydrogenation reaction to examine the involvement of the Y224 radical or Y68 radical in FtmOx1-catalysis, and again, the results from the verruculogen reaction are more consistent with the COX-like model.

OvoAMtht from Methyloversatilis thermotolerans ovothiol biosynthesis is a bifunction enzyme: thiol oxygenase and sulfoxide synthase activities.

Mononuclear non-heme iron enzymes are a large class of enzymes catalyzing a wide-range of reactions. In this work, we report that a non-heme iron enzyme in Methyloversatilis thermotolerans, OvoAMtht, has two different activities, as a thiol oxygenase and a sulfoxide synthase. When cysteine is presented as the only substrate, OvoAMtht is a thiol oxygenase. In the presence of both histidine and cysteine as substrates, OvoAMtht catalyzes the oxidative coupling between histidine and cysteine (a sulfoxide synthase). Additionally, we demonstrate that both substrates and the active site iron's secondary coordination shell residues exert exquisite control over the dual activities of OvoAMtht (sulfoxide synthase vs. thiol oxygenase activities). OvoAMtht is an excellent system for future detailed mechanistic investigation on how metal ligands and secondary coordination shell residues fine-tune the iron-center electronic properties to achieve different reactivities.

Light-driven Oxidative Demethylation Reaction Catalyzed by a Rieske-type Non-heme Iron Enzyme Stc2.

Rieske-type non-heme iron oxygenases/oxidases catalyze a wide range of transformations. Their applications in bioremediation or biocatalysis face two key barriers: the need of expensive NAD(P)H as a reductant and a proper reductase to mediate the electron transfer from NAD(P)H to the oxygenases. To bypass the need of both the reductase and NAD(P)H, using Rieske-type oxygenase (Stc2) catalyzed oxidative demethylation as the model system, we report Stc2 photocatalysis using eosin Y/sulfite as the photosensitizer/sacrificial reagent pair. In a flow-chemistry setting to separate the photo-reduction half-reaction and oxidation half-reaction, Stc2 photo-biocatalysis outperforms the Stc2-NAD(P)H-reductase (GbcB) system. In addition, in a few other selected Rieske enzymes (NdmA, CntA, and GbcA), and a flavin-dependent enzyme (iodotyrosine deiodinase, IYD), the eosin Y/sodium sulfite photo-reduction pair could also serve as the NAD(P)H-reductase surrogate to support catalysis, which implies the potential applicability of this photo-reduction system to other redox enzymes.

Molecular characterization of structural protein genes of dengue virus serotype 1 epidemic in Yunnan, Southwest China, in 2018.

A dengue virus serotype 1 (DENV-1) epidemic occurred from October to December 2018 in Xishuangbanna, Yunnan, Southwest China, neighboring Myanmar, Laos, and Vietnam. In this study, we investigated the molecular characteristics, evolution, and potential source of DENV from Xishuangbanna. The C (capsid), prM (premembrane), and E (envelope) genes of DENV isolated from 87 serum samples obtained from local patients were amplified and sequenced, and the sequences were evaluated by identification of mutations, phylogenetic and homologous recombination analysis, and secondary structure prediction. Phylogenetic analysis showed that all of the epidemic DENV strains from Xishuangbanna could be grouped in a branch with DENV-1 isolates, and were most similar to the Fujian 2005 (China, DQ193572) and Singapore 2016 (MF314188) strains. When compared with DENV-1SS (the standard strain), there were 31 non-synonymous mutations, but no obvious homologous recombination signal was found. Secondary structure prediction showed that some changes had occurred in a helical region in proteins of the MN123849 and MN123854 strains, but there were few changes in the disordered region. This study reveals the molecular characteristics of the structural genes of the Xishuangbanna epidemic strains in 2018 and provides a reference for molecular epidemiology, infection, and pathogenicity research and vaccine development.

Non-heme iron enzyme-catalyzed complex transformations: Endoperoxidation, cyclopropanation, orthoester, oxidative C-C and C-S bond formation reactions in natural product biosynthesis.

Non-heme iron enzymes catalyze a wide range of chemical transformations, serving as one of the key types of tailoring enzymes in the biosynthesis of natural products. Hydroxylation reaction is the most common type of reactions catalyzed by these enzymes and hydroxylation reactions have been extensively investigated mechanistically. However, the mechanistic details for other types of transformations remain largely unknown or unexplored. In this paper, we present some of the most recently discovered transformations, including endoperoxidation, orthoester formation, cyclopropanation, oxidative C-C and C-S bond formation reactions. In addition, many of them are multi-functional enzymes, which further complicate their mechanistic investigations. In this work, we summarize their biosynthetic pathways, with special emphasis on the mechanistic details available for these newly discovered enzymes.

Biochemical Characterization of a Multifunctional Mononuclear Nonheme Iron Enzyme (PtlD) in Neopentalenoketolactone Biosynthesis.

Pentalenolactone is a microbial sesquiterpenoid with antibiotic activity. Its biosynthetic pathway was elucidated by a combination of genetic and biochemical characterizations of all genes involved. For the related neopentalenoketolactone biosynthetic gene cluster from Streptomyces avermitilis, an α-ketoglutarate-dependent mononuclear nonheme iron enzyme, PtlD, was proposed to catalyze both desaturation and olefin epoxidation reactions. Yet, these activities remained to be validated by in vitro biochemical evidence. In this report, we demonstrated that PtlD has multiple activities, including hydroxylation, desaturation, and epoxidation, and confirmed the presence of the elusive epoxide intermediate in a neopentalenoketolactone pathway.

[Determination of fatty alkyl dimethyl tertiary amines by gas chromatography in the synthesis of cationic surfactants].

A method to determine fatty alkyl dimethyl tertiary amines by gas chromatography (GC) was set up using HP-INNOWax capillary column, hydrogen flame ionization detector (FID) and temperature programming. The linearities were all excellent in the range of 0.005-1.0 g/L with the correlation coefficients being above 0.9996. The limits of detection (LODs, S/N=3) of the method were between 0.001 g/L and 0.002 g/L, and the limits of quantification (LOQs, S/N=10) were between 0.003 g/L and 0.005 g/L. The recoveries ranged between 90% and 130% with relative standard deviations of 1.3%-6.9% (n=6). The proposed method has the advantages of wide linear range, higher recovery, and selectivity, which was suitable for the quantitative analysis of fatty alkyl dimethyl tertiary amines and monitoring process control in industrial production. The method was faster and more accurate than titration, and also precluded the need for pre-column derivatization and determination by liquid chromatography-tandem mass spectrometry.

In Vitro Reconstitution of the Remaining Steps in Ovothiol A Biosynthesis: C-S Lyase and Methyltransferase Reactions.

Ovothiols are thiolhistidine derivatives. The first step of ovothiol biosynthesis is OvoA-catalyzed oxidative coupling between histidine and cysteine. In this report, the remaining steps of ovothiol A biosynthesis were reconstituted in vitro. ETA_14770 (OvoB) was reported as a PLP-dependent sulfoxide lyase, responsible for mercaptohistidine production. OvoA was found to be a bifunctional enzyme, which mediates both oxidative C-S bond formation and methylation of mercaptohistidine to afford ovothiol A. Besides reconstituting the whole biosynthetic pathway, two unique features proposed in the literature were also examined: a potential cysteine-recycling mechanism of the C-S lyase (OvoB) and the selectivity of the π- N methyltransferase.

Recent examples of α-ketoglutarate-dependent mononuclear non-haem iron enzymes in natural product biosyntheses.

Covering: up to 2018 α-Ketoglutarate (αKG, also known as 2-oxoglutarate)-dependent mononuclear non-haem iron (αKG-NHFe) enzymes catalyze a wide range of biochemical reactions, including hydroxylation, ring fragmentation, C-C bond cleavage, epimerization, desaturation, endoperoxidation and heterocycle formation. These enzymes utilize iron(ii) as the metallo-cofactor and αKG as the co-substrate. Herein, we summarize several novel αKG-NHFe enzymes involved in natural product biosyntheses discovered in recent years, including halogenation reactions, amino acid modifications and tailoring reactions in the biosynthesis of terpenes, lipids, fatty acids and phosphonates. We also conducted a survey of the currently available structures of αKG-NHFe enzymes, in which αKG binds to the metallo-centre bidentately through either a proximal- or distal-type binding mode. Future structure-function and structure-reactivity relationship investigations will provide crucial information regarding how activities in this large class of enzymes have been fine-tuned in nature.

Snapshots of C-S Cleavage in Egt2 Reveals Substrate Specificity and Reaction Mechanism.

Sulfur incorporation in the biosynthesis of ergothioneine, a histidine thiol derivative, differs from other well-characterized transsulfurations. A combination of a mononuclear non-heme iron enzyme-catalyzed oxidative C-S bond formation and a subsequent pyridoxal 5'-phosphate (PLP)-mediated C-S lyase reaction leads to the net transfer of a sulfur atom from a cysteine to a histidine. In this study, we structurally and mechanistically characterized a PLP-dependent C-S lyase Egt2, which mediates the sulfoxide C-S bond cleavage in ergothioneine biosynthesis. A cation-π interaction between substrate and enzyme accounts for Egt2's preference of sulfoxide over thioether as a substrate. Using mutagenesis and structural biology, we captured three distinct states of the Egt2 C-S lyase reaction cycle, including a labile sulfenic intermediate captured in Egt2 crystals. Chemical trapping and high-resolution mass spectrometry were used to confirm the involvement of the sulfenic acid intermediate in Egt2 catalysis.

Use of a Tyrosine Analogue To Modulate the Two Activities of a Nonheme Iron Enzyme OvoA in Ovothiol Biosynthesis, Cysteine Oxidation versus Oxidative C-S Bond Formation.

Ovothiol is a histidine thiol derivative. The biosynthesis of ovothiol involves an extremely efficient trans-sulfuration strategy. The nonheme iron enzyme OvoA catalyzed oxidative coupling between cysteine and histidine is one of the key steps. Besides catalyzing the oxidative coupling between cysteine and histidine, OvoA also catalyzes the oxidation of cysteine to cysteine sulfinic acid (cysteine dioxygenase activity). Thus far, very little mechanistic information is available for OvoA-catalysis. In this report, we measured the kinetic isotope effect (KIE) in OvoA-catalysis using the isotopically sensitive branching method. In addition, by replacing an active site tyrosine (Tyr417) with 2-amino-3-(4-hydroxy-3-(methylthio)phenyl)propanoic acid (MtTyr) through the amber suppressor mediated unnatural amino acid incorporation method, the two OvoA activities (oxidative coupling between cysteine and histidine, and cysteine dioxygenase activity) can be modulated. These results suggest that the two OvoA activities branch out from a common intermediate and that the active site tyrosine residue plays some key roles in controlling the partitioning between these two pathways.

Tgfbr2 inactivation facilitates cellular plasticity and development of Pten-null prostate cancer.

Mutations in tumors can create a state of increased cellular plasticity that promotes resistance to treatment. Thus, there is an urgent need to develop novel strategies for identifying key factors that regulate cellular plasticity in order to combat resistance to chemotherapy and radiation treatment. Here we report that prostate epithelial cell reprogramming could be exploited to identify key factors required for promoting prostate cancer tumorigenesis and cellular plasticity. Deletion of phosphatase and tensin homolog (Pten) and transforming growth factor-beta receptor type 2 (Tgfbr2) may increase prostate epithelial cell reprogramming efficiency in vitro and cause rapid tumor development and early mortality in vivo. Tgfbr2 ablation abolished TGF-ß signaling but increased the bone morphogenetic protein (BMP) signaling pathway through the negative regulator Tmeff1. Furthermore, increased BMP signaling promotes expression of the tumor marker genes ID1, Oct4, Nanog, and Sox2; ID1/STAT3/NANOG expression was inversely correlated with patient survival. Thus, our findings provide information about the molecular mechanisms by which BMP signaling pathways render stemness capacity to prostate tumor cells.

USP26 functions as a negative regulator of cellular reprogramming by stabilising PRC1 complex components.

Despite much progress in the comprehension of the complex process of somatic cell reprogramming, many questions regarding the molecular mechanism of regulation remain to be answered. At present, the knowledge on the negative regulation of reprogramming process is indeed poor in contrary to the identification of positive regulators. Here we report for the first time that ubiquitin-specific protease 26 negatively regulates somatic cell-reprogramming process by stabilizing chromobox (CBX)-containing proteins CBX4 and CBX6 of polycomb-repressive complex 1 through the removal of K48-linked polyubiquitination. Thus, accumulated CBX4 and CBX6 repress the expression of pluripotency genes, such as Sox2 and Nanog, through PRC1 complexes to ubiquitinate histone H2A at their promoters. In all, our findings have revealed an essential role for ubiquitin-specific protease 26 in cellular reprogramming through polycomb-repressive complex 1.The ubiquitin-proteasome system regulates cellular reprogramming by degradation of key pluripotency factors. Here the authors report that the post-translational regulation of PRC1 components CBX4 and CBX6 by ubiquitination influences reprogramming.

Worldwide malaria incidence and cancer mortality are inversely associated.

BACKGROUND: Investigations on the effects of malaria infection on cancer mortality are limited except for the incidence of Burkitt's lymphoma (BL) in African children. Our previous murine lung cancer model study demonstrated that malaria infection significantly inhibited tumor growth and prolonged the life span of tumor-bearing mice. This study aims to assess the possible associations between malaria incidence and human cancer mortality. METHODS: We compiled data on worldwide malaria incidence and age-standardized mortality related to 30 types of cancer in 56 countries for the period 1955-2008, and analyzed their longitudinal correlations by a generalized additive mixed model (GAMM), adjusted for a nonlinear year effect and potential confounders such as country's income levels, life expectancies and geographical locations. RESULTS: Malaria incidence was negatively correlated with all-cause cancer mortality, yielding regression coefficients (log scale) of -0.020 (95%CI: -0.027,-0.014) for men (P < 0.001) and-0.020 (95%CI: -0.025,-0.014) for women (P < 0.001). Among the 29 individual types of cancer studied, malaria incidence was negatively correlated with colorectum and anus (men and women), colon (men and women), lung (men), stomach (men), and breast (women) cancer. CONCLUSIONS: Our analysis revealed a possible inverse association between malaria incidence and the mortalities of all-cause and some types of solid cancers, which is opposite to the known effect of malaria on the pathogenesis of Burkitt's lymphoma. Activation of the whole immune system, inhibition of tumor angiogenesis by Plasmodium infection may partially explain why endemic malaria might reduce cancer mortality at the population level.

Protein arginine methyltransferase 1 may be involved in pregnane x receptor-activated overexpression of multidrug resistance 1 gene during acquired multidrug resistant.

PURPOSE: Pregnane x receptor (PXR) - activated overexpression of the multidrug resistance 1 (MDR1) gene is an important way for tumor cells to acquire drug resistance. However, the detailed mechanism still remains unclear. In the present study, we aimed to investigate whether protein arginine methyl transferase 1(PRMT1) is involved in PXR - activated overexpression of MDR1 during acquired multidrug resistant. EXPERIMENTAL DESIGN: Arginine methyltransferase inhibitor 1 (AMI-1) was used to pharmacologically block PRMT1 in resistant breast cancer cells (MCF7/adr). The mRNA and protein levels of MDR1 were detected by real-time PCR and western blotting analysis. Immunofluorescence microscopy and co-immunoprecipitation were used to investigate the physical interaction between PXR and PRMT1. Then, 136 candidate compounds were screened for PRMT1 inhibitors. Lastly, luciferase reporter gene and nude mice bearing resistant breast cancer xenografts were adopted to investigate the anti-tumor effect of PRMT1 inhibitors when combined with adriamycin. RESULTS: AMI-1 significantly suppressed the expression of MDR1 in MCF7/adr cells and increased cells sensitivity of MCF7/adr to adriamycin. Physical interaction between PRMT1 and PXR exists in MCF7/adr cells, which could be disrupted by AMI-1. Those results suggest that PRMT1 may be involved in PXR-activated overexpression of MDR1 in resistant breast cancer cells, and AMI-1 may suppress MDR1 by disrupting the interaction between PRMT1 and PXR. Then, five compounds including rutin, isoquercitrin, salvianolic acid A, naproxen, and felodipline were identified to be PRMT1 inhibitors. Finally, those PRMT1 inhibitors were observed to significantly decrease MDR1 promoter activity in vitro and enhance the antitumor effect of adriamycin in nude mice that bearing resistant breast cancer xenografts. CONCLUSIONS: PRMT1 may be an important co-activator of PXR in activating MDR1 gene during acquired resistance, and PRMT1 inhibitor combined with chemotherapy drugs may be a new strategy for overcoming tumor MDR.

Endoperoxide formation by an α-ketoglutarate-dependent mononuclear non-haem iron enzyme.

Many peroxy-containing secondary metabolites have been isolated and shown to provide beneficial effects to human health. Yet, the mechanisms of most endoperoxide biosyntheses are not well understood. Although endoperoxides have been suggested as key reaction intermediates in several cases, the only well-characterized endoperoxide biosynthetic enzyme is prostaglandin H synthase, a haem-containing enzyme. Fumitremorgin B endoperoxidase (FtmOx1) from Aspergillus fumigatus is the first reported α-ketoglutarate-dependent mononuclear non-haem iron enzyme that can catalyse an endoperoxide formation reaction. To elucidate the mechanistic details for this unique chemical transformation, we report the X-ray crystal structures of FtmOx1 and the binary complexes it forms with either the co-substrate (α-ketoglutarate) or the substrate (fumitremorgin B). Uniquely, after α-ketoglutarate has bound to the mononuclear iron centre in a bidentate fashion, the remaining open site for oxygen binding and activation is shielded from the substrate or the solvent by a tyrosine residue (Y224). Upon replacing Y224 with alanine or phenylalanine, the FtmOx1 catalysis diverts from endoperoxide formation to the more commonly observed hydroxylation. Subsequent characterizations by a combination of stopped-flow optical absorption spectroscopy and freeze-quench electron paramagnetic resonance spectroscopy support the presence of transient radical species in FtmOx1 catalysis. Our results help to unravel the novel mechanism for this endoperoxide formation reaction.

Mechanistic studies of a novel C-S lyase in ergothioneine biosynthesis: the involvement of a sulfenic acid intermediate.

Ergothioneine is a histidine thio-derivative isolated in 1909. In ergothioneine biosynthesis, the combination of a mononuclear non-heme iron enzyme catalyzed oxidative C-S bond formation reaction and a PLP-mediated C-S lyase (EgtE) reaction results in a net sulfur transfer from cysteine to histidine side-chain. This demonstrates a new sulfur transfer strategy in the biosynthesis of sulfur-containing natural products. Due to difficulties associated with the overexpression of Mycobacterium smegmatis EgtE protein, the proposed EgtE functionality remained to be verified biochemically. In this study, we have successfully overexpressed and purified M. smegmatis EgtE enzyme and evaluated its activities under different in vitro conditions: C-S lyase reaction using either thioether or sulfoxide as a substrate in the presence or absence of reductants. Results from our biochemical characterizations support the assignment of sulfoxide 4 as the native EgtE substrate and the involvement of a sulfenic acid intermediate in the ergothioneine C-S lyase reaction.