In a conventional class I ribonucleotide reductase (RNR), a diiron(II/II) cofactor in the R2 subunit reacts with oxygen to produce a diiron(III/IV) intermediate, which generates a stable tyrosyl radical (Y*). The Y* reversibly oxidizes a cysteine residue in the R1 subunit to a cysteinyl radical (C*), which abstracts the 3'-hydrogen of the substrate to initiate its reduction. The RNR from Chlamydia trachomatis lacks the Y*, and it had been proposed that the diiron(III/IV) complex in R2 directly generates the C* in R1. By enzyme activity measurements and spectroscopic methods, we show that this RNR actually uses a previously unknown stable manganese(IV)/iron(III) cofactor for radical initiation.

. Recent studies have suggested that the enzyme, which was shown nearly 50 years ago to require iron (1, 2), contains a coupled dinuclear nonheme iron cluster (5), making MIOX the most recent addition to the nonheme diiron oxygenase͞oxidase family that also includes bacterial hydrocarbon hydroxylases (e.g., soluble methane monooxygenase), plant fatty acyl desaturases (e.g., stearoyl acyl carrier protein ⌬ 9 desaturase), and protein R2 of class I ribonucleotide reductase (R2) (6-10). Mössbauer and EPR spectra showed that treatment of recombinant Mus musculus MIOX isolated in its iron-free form from Escherichia coli with Fe(II) and O 2 leads to formation of an antiferromagnetically coupled diiron cluster in either the II͞III or III͞III oxidation state, depending on the O 2 ͞MIOX ratio and the presence or absence of a reductant (ascorbate or cysteine). Binding of MI was shown to perturb the spectra of both oxidation states in a manner consistent with direct coordination of the substrate to the cluster (5).All nonheme diiron oxygenases and oxidases characterized before MIOX activate O 2 with the II͞II oxidation state of the cofactor (11,12). For several of the reactions, (peroxo)diiron(III͞III) intermediates have been demonstrated. These complexes are generally proposed to undergo O-O-bond cleavage to generate high-valent iron complexes that cleave strong C-H or O-H bonds of their oxidation targets (8,(11)(12)(13)(14). Indeed, the diiron(III͞IV) cluster, X (15, 16), and the diiron(IV͞IV) cluster, Q (8, 13, 17), have been directly characterized in the R2 and soluble methane monooxygenase reactions, respectively. In each of the previously characterized diiron-oxygenase͞oxidase reactions, a diiron(III͞III) ''product'' state of the cluster is generated at the end of the oxidation sequence. Subsequent events require reduction of the cluster back to the diiron(II͞II) state by additional proteins, with electrons provided ultimately by NAD(P)H. This redox cycling of the cofactor and provision of two electrons by the nicotinamide ''cosubstrate'' ensure that at most two electrons can be extracted from the substrate. The MIOX reaction, a four-electron oxidation, would seem to require a different mechanism.Indeed, a recent study concluded that the mixed-valent, II͞III state of the cofactor, rather than the conventional II͞II state, activates O 2 for DG production in the MIOX reaction (4). Single-turnover experiments showed that the fully reduced enzyme (MIOX II/II ) reacts with limiting O 2 in the presence of saturating MI to generate the mixed-valent enzyme as a stable product with unit stoichiometry and with only a low yield of DG. By contrast, the

The enzyme isopenicillin N synthase (IPNS) installs the β-lactam and thiazolidine rings of the penicillin core into the linear tripeptide, L-δ-aminoadipoyl-L-Cys-D-Val (ACV), on the pathways to a number of important antibacterial drugs. A classic set of enzymological and crystallographic studies by Baldwin and co-workers established that this overall four-electron oxidation occurs by a sequence of two oxidative cyclizations, with the β-lactam ring being installed first and the thiazolidine ring second. Each phase requires cleavage of an aliphatic C–H bond of the substrate: the pro-S-CCys,β-H bond for closure of the β-lactam ring, and the CVal,β-H bond for installation of the thiazolidine ring. IPNS uses a mononuclear non-heme-iron(II) cofactor and dioxygen as co-substrate to cleave these C–H bonds and direct the ring closures. Despite the intense scrutiny to which the enzyme has been subjected, the identities of the oxidized iron intermediates that cleave the C–H bonds have been addressed only computationally; no experimental insight into their geometric or electronic structures has been reported. In this work, we have employed a combination of transient-state-kinetic and spectroscopic methods, together with the specifically deuterium-labeled substrates, A[d2-C]V and AC[d8-V], to identify both C–H-cleaving intermediates. The results show that they are high-spin Fe(III)-superoxo and high-spin Fe(IV)-oxo complexes, respectively, in agreement with published mechanistic proposals derived computationally from Baldwin’s founding work.

myo-Inositol oxygenase (MIOX) uses iron as its cofactor and dioxygen as its cosubstrate to effect the unique, ring-cleaving, four-electron oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate to d-glucuronate. The nature of the iron cofactor and its interaction with the substrate, myo-inositol (MI), have been probed by electron paramagnetic resonance (EPR) and Mössbauer spectroscopies. The data demonstrate the formation of an antiferromagnetically coupled, high-spin diiron(III/III) cluster upon treatment of solutions of Fe(II) and MIOX with excess O(2) or H(2)O(2) and the formation of an antiferromagnetically coupled, valence-localized, high-spin diiron(II/III) cluster upon treatment with either limiting O(2) or excess O(2) in the presence of a mild reductant (e.g., ascorbate). Marked changes to the spectra of both redox forms upon addition of MI and analogy to changes induced by binding of phosphate to the diiron(II/III) cluster of the protein phosphatase, uteroferrin, suggest that MI coordinates directly to the diiron cluster, most likely in a bridging mode. The addition of MIOX to the growing family of non-heme diiron oxygenases expands the catalytic range of the family beyond the two-electron oxidation (hydroxylation and dehydrogenation) reactions catalyzed by its more extensively studied members such as methane monooxygenase and stearoyl acyl carrier protein Delta(9)-desaturase.

Mammalian target of rapamycin (mTOR) regulates various cellular functions, including tumorigenesis, and is inhibited by the tuberous sclerosis 1 (TSC1)–TSC2 complex. Here, we demonstrate that arrest-defective protein 1 (ARD1) physically interacts with, acetylates, and stabilizes TSC2, thereby repressing mTOR activity. The inhibition of mTOR by ARD1 inhibits cell proliferation and increases autophagy, thereby inhibiting tumorigenicity. Correlation between ARD1 and TSC2 abundance was apparent in multiple tumor types. Moreover, evaluation of loss of heterozygosity at Xq28 revealed allelic loss in 31% of tested breast cancer cell lines and tumor samples. Together, our findings suggest that ARD1 functions as an inhibitor of the mTOR pathway and that dysregulation of the ARD1-TSC2-mTOR axis may contribute to cancer development.

Porcine circovirus 3 (PCV3) is a novel virus associated with acute PDNS (porcine dermatitis and nephropathy syndrome)‐like clinical signs identified by metagenomic sequencing from swine. Its high occurrence may pose a potential threat to the swine industry worldwide. The processes resulting in the emergence and spread of PCV3 remain poorly understood. Herein, the possible origin, genotypes, and evolutionary dynamics of PCV3 based on available genomic sequences are determined. The closest ancestor of PCV3 is found to be within the clade 1 bat CVs. Using different phylogenetic methods, two major genotypes are identified, PCV3a and PCV3b. It is found that the effective population size of PCV3 increased rapidly during late 2013 to early 2014 and this is associated with the diversification of PCV3a and PCV3b. A relatively high effective reproductive number (Re) value and higher evolutionary rate were found compared to other single‐stranded DNA viruses, and positive selection on codons 122 and 320 (24 of ORF2) is identified. It is hypothesized that this, together with the prediction of a potential change of an antigenic epitope at position 320, might have allowed PCV3 to escape from the host immune response. Overall, this study has important implications for understanding the ongoing PCV3 cases worldwide and will guide future efforts to develop effective preventive and control measures.

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The enzyme myo-inositol oxygenase (MIOX) catalyzes conversion of myo-inositol (cyclohexan-1,2,3,5/4,6-hexa-ol or MI) to D-glucuronate (DG), initiating the only known pathway in humans for catabolism of the carbon skeleton of cell-signaling inositol (poly)phosphates and phosphoinositides. Recent kinetic, spectroscopic, and crystallographic studies have shown that the enzyme activates its substrates, MI and O 2 , at a carboxylate-bridged nonheme diiron(II/III) cluster, making it the first of many known nonheme diiron oxygenases to employ the mixed-valent form of its cofactor. Evidence suggests that (1) the Fe(III) site coordinates MI via its C1 and C6 hydroxyl groups, (2) the Fe(II) site reversibly coordinates O 2 to produce a superoxo-diiron(III/III) intermediate, and (3) the pendant oxygen atom of the superoxide ligand abstracts hydrogen from C1 to initiate the unique C-C-bond-cleaving, four-electron oxidation reaction. This review recounts the studies leading to the recognition of the novel cofactor requirement and catalytic mechanism of MIOX and forecasts how remaining gaps in our understanding might be filled by additional experiments.Bacterial multi-component monooxygenases [BMMs; e.g., soluble methane monooxygenase (sMMO), toluene/o-xylene monooxygenase (ToMO), and phenol hydroxylase], plant fatty acyl desaturases (e.g., stearoyl acyl carrier protein Δ 9 -desaturase) and the R2 subunits of conventional class I ribonucleotide reductases (RNR-R2s) all use carboxylate-bridged dinuclear iron clusters to activate O 2 for cleavage of strong C-H or O-H bonds. 1-5 Each of these reaction begins with the reduction of O 2 to the peroxide oxidation state by the diiron(II/ II) form of the cofactor. In several cases, peroxide-bridged diiron(III/III) intermediates have been directly characterized. 6-11 Several of the peroxide complexes are known or believed to undergo O-O-bond cleavage to generate high-valent iron complexes that cleave the target C/ O-H bonds. 1-5 For example, the diiron(III/IV) complex, X, in the RNR-R2 reaction oxidizes a tyrosine residue by one electron, cleaving the phenolic O-H bond and activating the protein for participation in nucleotide reduction with its partner subunit, RNR-R1. [12][13][14][15][16][17][18][19][20][21] Similarly, the diiron(IV/IV) complex, Q, in the sMMO reaction cleaves a C-H bond of methane to initiate its hydroxylation. 1,3,7,[22][23][24] In each of these reactions, a diiron(III/III) form of the cluster is generated at the end of the oxidation sequence. For the reactions that are catalytic, a complete "turnover" therefore requires reduction of the cluster back to the O 2 -reactive diiron(II/II) state by additional proteins, with electrons provided ultimately by NAD(P)H. 1,3,4 Although this Please send correspondence to: J.