45 research outputs found
Kinetic mechanism of ornithine hydroxylase (PvdA) from Pseudomonas aeruginosa: substrate triggering of O2 addition but not flavin reduction
This publication was made possible by NIH Grant P20 RR-17708-05 from the National Center for Research Resources of the National Institutes of Health. K.M.M. was a recipient of a National Institutes of Health Predoctoral Training Grant Fellowship (GM08545).PvdA catalyzes the hydroxylation of the sidechain primary amine of ornithine in the initial step of the biosynthesis of the Pseudomonas aeruginosa siderophore pyoverdin. The reaction requires FAD, NADPH, and O2. PvdA uses the same co-substrates as several flavin-dependent hydroxylases that differ one from another in the kinetic mechanisms of their oxidative and reductive half-reactions. Therefore, the mechanism of PvdA was determined by absorption stopped-flow experiments. By contrast to some flavin-dependent hydroxylases (notably, p-hydroxybenzoate hydroxylase), binding of the hydroxylation target is not required to trigger reduction of the flavin by NADPH: the reductive half-reaction is equally facile in the presence and absence of ornithine. Reaction of O2 with FADH2 in the oxidative half-reaction is accelerated by ornithine 80-fold, providing a mechanism by which PvdA can ensure coupling of NADPH and ornithine oxidation. In the presence of ornithine, the expected C(4a)-hydroperoxyflavin intermediate with 390-nm absorption accumulates and decays to the C(4a)-hydroxyflavin in a kinetically competent fashion. The slower oxidative half-reaction that occurs in the absence of ornithine involves accumulation of an oxygenated flavin species and two subsequent states that are tentatively assigned as C(4a)-peroxy- and -hydroperoxyflavin intermediates and the oxidized flavin. The enzyme generates stoichiometric hydrogen peroxide in lieu of hydroxyornithine. The data suggest that PvdA employs a kinetic mechanism that is a hybrid of those previously documented for other flavin-dependent hydroxylases
Lethal Mutagenesis of Picornaviruses with N-6-Modified Purine Nucleoside Analogues
RNA viruses exhibit extraordinarily high mutation rates during genome replication. Nonnatural ribonucleosides that can increase the mutation rate of RNA viruses by acting as ambiguous substrates during replication have been explored as antiviral agents acting through lethal mutagenesis. We have synthesized novel N-6-substituted purine analogues with ambiguous incorporation characteristics due to tautomerization of the nucleobase. The most potent of these analogues reduced the titer of poliovirus (PV) and coxsackievirus (CVB3) over 1,000-fold during a single passage in HeLa cell culture, with an increase in transition mutation frequency up to 65-fold. Kinetic analysis of incorporation by the PV polymerase indicated that these analogues were templated ambiguously with increased efficiency compared to the known mutagenic nucleoside ribavirin. Notably, these nucleosides were not efficient substrates for cellular ribonucleotide reductase in vitro, suggesting that conversion to the deoxyriboucleoside may be hindered, potentially limiting genetic damage to the host cell. Furthermore, a high-fidelity PV variant (G64S) displayed resistance to the antiviral effect and mutagenic potential of these analogues. These purine nucleoside analogues represent promising lead compounds in the development of clinically useful antiviral therapies based on the strategy of lethal mutagenesis
Hydrogen Donation but not Abstraction by a Tyrosine (Y68) During Endoperoxide Installation by Verruculogen Synthase (FtmOx1)
Hydrogen-atom transfer (HAT) from a substrate carbon to an iron(IV)-oxo (ferryl) intermediate initiates a diverse array of enzymatic transformations. For outcomes other than hydroxylation, coupling of the resultant carbon radical and hydroxo ligand (oxygen rebound) must generally be averted. A recent study of FtmOx1, a fungal iron(II)- and 2-(oxo)glutarate-dependent oxygenase that installs the endoperoxide of verruculogen by adding O_2 between carbons 21 and 27 of fumitremorgin B, posited that tyrosine (Tyr or Y) 224 serves as HAT intermediary to separate the C21 radical (C21•) and Fe(III)–OH HAT products and prevent rebound. Our reinvestigation of the FtmOx1 mechanism revealed, instead, direct HAT from C21 to the ferryl complex and surprisingly competitive rebound. The C21-hydroxylated (rebound) product, which undergoes deprenylation, predominates when low [O_2] slows C21•–O_2 coupling in the next step of the endoperoxidation pathway. This pathway culminates with addition of the C21–O–O• peroxyl adduct to olefinic C27 followed by HAT to the C26• from a Tyr. The last step results in sequential accumulation of Tyr radicals, which are suppressed without detriment to turnover by inclusion of the reductant, ascorbate. Replacement of each of four candidates for the proximal C26 H• donor (including Y224) with phenylalanine (F) revealed that only the Y68F variant (i) fails to accumulate the first Tyr• and (ii) makes an altered major product, identifying Y68 as the donor. The implied proximities of C21 to the iron cofactor and C26 to Y68 support a new docking model of the enzyme–substrate complex that is consistent with all available data
Modern tests of Lorentz invariance
Motivated by ideas about quantum gravity, a tremendous amount of effort over
the past decade has gone into testing Lorentz invariance in various regimes.
This review summarizes both the theoretical frameworks for tests of Lorentz
invariance and experimental advances that have made new high precision tests
possible. The current constraints on Lorentz violating effects from both
terrestrial experiments and astrophysical observations are presented.Comment: Modified and expanded discussions of various points. Numerous
references added. Version matches that accepted by Living Reviews in
Relativit
Transcriptome of Aphanomyces euteiches: New Oomycete Putative Pathogenicity Factors and Metabolic Pathways
Aphanomyces euteiches is an oomycete pathogen that causes seedling blight and root rot of legumes, such as alfalfa and pea. The genus Aphanomyces is phylogenically distinct from well-studied oomycetes such as Phytophthora sp., and contains species pathogenic on plants and aquatic animals. To provide the first foray into gene diversity of A. euteiches, two cDNA libraries were constructed using mRNA extracted from mycelium grown in an artificial liquid medium or in contact to plant roots. A unigene set of 7,977 sequences was obtained from 18,864 high-quality expressed sequenced tags (ESTs) and characterized for potential functions. Comparisons with oomycete proteomes revealed major differences between the gene content of A. euteiches and those of Phytophthora species, leading to the identification of biosynthetic pathways absent in Phytophthora, of new putative pathogenicity genes and of expansion of gene families encoding extracellular proteins, notably different classes of proteases. Among the genes specific of A. euteiches are members of a new family of extracellular proteins putatively involved in adhesion, containing up to four protein domains similar to fungal cellulose binding domains. Comparison of A. euteiches sequences with proteomes of fully sequenced eukaryotic pathogens, including fungi, apicomplexa and trypanosomatids, allowed the identification of A. euteiches genes with close orthologs in these microorganisms but absent in other oomycetes sequenced so far, notably transporters and non-ribosomal peptide synthetases, and suggests the presence of a defense mechanism against oxidative stress which was initially characterized in the pathogenic trypanosomatids
The self-organizing fractal theory as a universal discovery method: the phenomenon of life
A universal discovery method potentially applicable to all disciplines studying organizational phenomena has been developed. This method takes advantage of a new form of global symmetry, namely, scale-invariance of self-organizational dynamics of energy/matter at all levels of organizational hierarchy, from elementary particles through cells and organisms to the Universe as a whole. The method is based on an alternative conceptualization of physical reality postulating that the energy/matter comprising the Universe is far from equilibrium, that it exists as a flow, and that it develops via self-organization in accordance with the empirical laws of nonequilibrium thermodynamics. It is postulated that the energy/matter flowing through and comprising the Universe evolves as a multiscale, self-similar structure-process, i.e., as a self-organizing fractal. This means that certain organizational structures and processes are scale-invariant and are reproduced at all levels of the organizational hierarchy. Being a form of symmetry, scale-invariance naturally lends itself to a new discovery method that allows for the deduction of missing information by comparing scale-invariant organizational patterns across different levels of the organizational hierarchy
Rapid-Freeze-Quench Magnetic Circular Dichroism of Intermediate X in Ribonucleotide Reductase: New Structural Insight
To elucidate the electronic structure of intermediate X in the oxygen activation reaction of the R2 subunit of ribonucleotide reductase, a protocol has been developed to perform magnetic circular dichroism (MCD) on a rapid-freeze-quench, strain free optical sample. RFQ-MCD data have been collected on intermediate X in the double mutant of R2, Y122/Y356F. While X has been reported to exhibit a broad absorption band at 365 nm, there are at least 10 electronic transitions observed at low-temperature MCD. From C0/D0 ratios, the transitions of X can be divided into three regions: 16 000-22 000 cm-1 region involving spin-allowed ligand field transitions of the Fe(IV), 23 000-24 000 cm-1 region of spin-forbidden, spin-flip transitions on the Fe(IV), and the charge transfer (CT) region from 26 000 to 32 000 cm-1. The C0/D0 ratios from d --> d and CT transitions strongly support significant Fe(IV) character coupled into the paramagnetic center. Ligand field (spin-allowed d --> d region) analysis allows the bis-mu-oxo and mu-oxo plus other monoanionic bridge possibilities for the structure of intermediate X to be distinguished, providing new insight into the molecular mechanism of the cluster formation in R2
Rapid-freeze-quench magnetic circular dichroism of intermediate X in ribonucleotide reductase: New structural insight
To elucidate the electronic structure of intermediate X in the oxygen activation reaction of the R2 subunit of ribonucleotide reductase, a protocol has been developed to perform magnetic circular dichroism (MCD) on a rapid-freeze-quench, strain free optical sample. RFQ-MCD data have been collected on intermediate X in the double mutant of R2, Y122/Y356F. While X has been reported to exhibit a broad absorption band at 365 nm, there are at least 10 electronic transitions observed at low-temperature MCD. From C0/D0 ratios, the transitions of X can be divided into three regions: 16 000-22 000 cm-1 region involving spin-allowed ligand field transitions of the Fe(IV), 23 000-24 000 cm-1 region of spin-forbidden, spin-flip transitions on the Fe(IV), and the charge transfer (CT) region from 26 000 to 32 000 cm-1. The C0/D0 ratios from d --> d and CT transitions strongly support significant Fe(IV) character coupled into the paramagnetic center. Ligand field (spin-allowed d --> d region) analysis allows the bis-mu-oxo and mu-oxo plus other monoanionic bridge possibilities for the structure of intermediate X to be distinguished, providing new insight into the molecular mechanism of the cluster formation in R2
Spectroscopic and electronic structure studies of intermediate X in ribonucleotide reductase R2 and two variants: a description of the Fe IV-oxo bond in the FeIII-O-FeIV dimer
Spectroscopic and electronic structure studies of the class I Escherichia coli ribonucleotide reductase (RNR) intermediate X and three computationally derived model complexes are presented, compared, and evaluated to determine the electronic and geometric structure of the Fe-Fe active site of intermediate X. Rapid freeze-quench (RFQ) EPR, absorption, and MCD were used to trap intermediate X in R2 wild-type (WT) and two variants, W48A and Y122F/Y356F. RFQ-EPR spin quantitation was used to determine the relative contributions of intermediate X and radicals present, while RFQ-MCD was used to specifically probe the Fe/Fe active site, which displayed three Fe d-d transitions between 16 700 and 22 600 cm, two Fe d-d spin-flip transitions between 23 500 and 24 300 cm, and five oxo to Fe and Fe charge transfer (CT) transitions between 25 000 and 32 000 cm. The Fe d-d transitions were perturbed in the two variants, confirming that all three d-d transitions derive from the d-π manifold. Furthermore, the Fe d-π splittings in the WT are too large to correlate with a bis-μ-oxo structure. The assignment of the Fe d-d transitions in WT intermediate X best correlates with a bridged μ-oxo/μ-hydroxo [Fe (μ-O)(μ-OH)Fe] structure. The μ-oxo/μ-hydroxo core structure provides an important σ/π superexchange pathway, which is not present in the bis-μ-oxo structure, to promote facile electron transfer from Y122 to the remote Fe through the bent oxo bridge, thereby generating the tyrosyl radical for catalysis