Initial Steps in Methanobactin Biosynthesis: Substrate Binding by the Mixed-Valent Diiron Enzyme MbnBC.

The MbnBC enzyme complex converts cysteine residues in a peptide substrate, MbnA, to oxazolone/thioamide groups during the biosynthesis of copper chelator methanobactin (Mbn). MbnBC belongs to the mixed-valent diiron oxygenase (MVDO) family, of which members use an Fe(II)Fe(III) cofactor to react with dioxygen for substrate modification. Several crystal structures of the inactive Fe(III)Fe(III) form of MbnBC alone and in complex with MbnA have been reported, but a mechanistic understanding requires determination of the oxidation states of the crystallographically observed Fe ions in the catalytically active Fe(II)Fe(III) state, along with the site of MbnA binding. Here, we have used electron nuclear double resonance (ENDOR) spectroscopy to determine such structural and electronic properties of the active site, in particular, the mode of substrate binding to the MV state, information not accessible by X-ray crystallography alone. The oxidation states of the two Fe ions were determined by 15N ENDOR analysis. The presence and locations of both bridging and terminal exogenous solvent ligands were determined using 1H and 2H ENDOR. In addition, 2H ENDOR using an isotopically labeled MbnA substrate indicates that MbnA binds to the Fe(III) ion of the cluster via the sulfur atom of its N-terminal modifiable cysteine residue, with displacement of a coordinated solvent ligand as shown by complementary 1H ENDOR. These results, which underscore the utility of ENDOR in studying MVDOs, provide a molecular picture of the initial steps in Mbn biosynthesis.

Integrative analysis of metabolite and transcriptome reveals biosynthetic pathway and candidate genes for eupatilin and jaceosidin biosynthesis in Artemisia argyi.

Artemisia argyi (A. argyi) is a medicinal plant belonging to the Asteraceae family and Artemisia genus. Flavonoids abundant in A. argyi are associated with anti-inflammatory, anticancer, and antioxidative effects. Eupatilin and jaceosidin are representative polymethoxy flavonoids with medicinal properties significant enough to warrant the development of drugs using their components. However, the biosynthetic pathways and related genes of these compounds have not been fully explored in A. argyi. This study comprehensively analyzed the transcriptome data and flavonoids contents from four different tissues of A. argyi (young leaves, old leaves, trichomes collected from stems, and stems without trichomes) for the first time. We obtained 41,398 unigenes through the de-novo assembly of transcriptome data and mined promising candidate genes involved in the biosynthesis of eupatilin and jaceosidin using differentially expressed genes, hierarchical clustering, phylogenetic tree, and weighted gene co-expression analysis. Our analysis led to the identification of a total of 7,265 DEGs, among which 153 genes were annotated as flavonoid-related genes. In particular, we were able to identify eight putative flavone-6-hydroxylase (F6H) genes, which were responsible for providing a methyl group acceptor into flavone basic skeleton. Furthermore, five O-methyltransferases (OMTs) gene were identified, which were required for the site-specific O-methylation during the biosynthesis of eupatilin and jaceosidin. Although further validation would be necessary, our findings pave the way for the modification and mass-production of pharmacologically important polymethoxy flavonoids through genetic engineering and synthetic biological approaches.

A mixed-valent Fe(II)Fe(III) species converts cysteine to an oxazolone/thioamide pair in methanobactin biosynthesis.

SignificanceMethanobactins (Mbns), copper-binding peptidic compounds produced by some bacteria, are candidate therapeutics for human diseases of copper overload. The paired oxazolone-thioamide bidentate ligands of methanobactins are generated from cysteine residues in a precursor peptide, MbnA, by the MbnBC enzyme complex. MbnBC activity depends on the presence of iron and oxygen, but the catalytically active form has not been identified. Here, we provide evidence that a dinuclear Fe(II)Fe(III) center in MbnB, which is the only representative of a >13,000-member protein family to be characterized, is responsible for this reaction. These findings expand the known roles of diiron enzymes in biology and set the stage for mechanistic understanding, and ultimately engineering, of the MbnBC biosynthetic complex.

Characterization of a Copper-Chelating Natural Product from the Methanotroph Methylosinus sp. LW3.

Methanobactins (Mbns) are ribosomally produced, post-translationally modified peptidic natural products that bind copper with high affinity. Methanotrophic bacteria use Mbns to acquire copper needed for enzymatic methane oxidation. Despite the presence of Mbn operons in a range of methanotroph and other bacterial genomes, few Mbns have been isolated and structurally characterized. Here we report the isolation of a novel Mbn from the methanotroph Methylosinus (Ms.) sp. LW3. Mass spectrometric and nuclear magnetic resonance spectroscopic data indicate that this Mbn, the largest characterized to date, consists of a 13-amino acid backbone modified to include pyrazinedione/oxazolone rings and neighboring thioamide groups derived from cysteine residues. The pyrazinedione ring is more stable to acid hydrolysis than the oxazolone ring and likely protects the Mbn from degradation. The structure corresponds exactly to that predicted on the basis of the Ms. sp. LW3 Mbn operon content, providing support for the proposed role of an uncharacterized biosynthetic enzyme, MbnF, and expanding the diversity of known Mbns.

A bioinspired iron catalyst for nitrate and perchlorate reduction.

Nitrate and perchlorate have considerable use in technology, synthetic materials, and agriculture; as a result, they have become pervasive water pollutants. Industrial strategies to chemically reduce these oxyanions often require the use of harsh conditions, but microorganisms can efficiently reduce them enzymatically. We developed an iron catalyst inspired by the active sites of nitrate reductase and (per)chlorate reductase enzymes. The catalyst features a secondary coordination sphere that aids in oxyanion deoxygenation. Upon reduction of the oxyanions, an iron(III)-oxo is formed, which in the presence of protons and electrons regenerates the catalyst and releases water.

Repeated occurrence of second primary lung cancer at different sites in trachea: a case report.

Multiple or second primary lung cancers can develop at any sites in the lung with same or different histologic types, synchronously and/or metachronously. In case of metachronous occurrence of the second primary lung cancer, it is easy to confuse with the primary lung cancer as a recurrence of precedent lung malignancy treated successfully or metastasis. Previous reports have demonstrated that majority of the second primary lung malignancies have same histologic types regardless of their developing time and location. However, the repeated occurrence of the second primary lung malignancy, in particular with the different histologic features, is a very rare condition.A 62-year-old male who had past history of squamous cell carcinoma treated with surgery and adjuvant chemotherapy and the recurrence of lung malignancy on the trachea, which was also resected successfully visited our hospital due to blood tinged sputum. Evaluation using bronchoscopy and chest computed tomography revealed the tracheal mass looked similar grossly to the previous recurred tracheal mass that was resected surgically. Unexpectedly, the newly developed tracheal mass was confirmed as small cell lung cancer, the different histologic type from previous ones.In this report, we describe an interesting case of subsequent occurrence of second primary lung cancers showing histologic shifting at different sites in trachea, suggesting that it is important for physician to make an effort to identify the histologic characteristics of second primary lung cancers for the correct and adequate treatment no matter what they exhibit similar gross morphology.

Facile nitrite reduction in a non-heme iron system: formation of an iron(III)-oxo.

Reaction of tetrabutylammonium nitrite with [N(afa(Cy))3Fe(OTf)](OTf) cleanly resulted in the formation of an iron(III)-oxo species, [N(afa(Cy))3Fe(O)](OTf), and NO(g). Formation of NO(g) as a byproduct was confirmed by reaction of the iron(II) starting material with half an equivalent of nitrite, resulting in a mixture of two products, the iron-oxo and an iron-NO species, [N(afa(Cy))3Fe(NO)](OTf)2. Formation of the latter was confirmed through independent synthesis. The results of this study provide insight into the role of hydrogen bonding in the mechanism of nitrite reduction and the binding mode of nitrite in biological heme systems.