Crystal structure of the RNA polymerase domain of the West Nile Virus non-structural protein 5

Viruses of the family Flaviviridae are important human and animal pathogens. Among them, the Flaviviruses dengue ( DENV) and West Nile ( WNV) cause regular outbreaks with fatal outcomes. The RNA-dependent RNA polymerase ( RdRp) activity of the non-structural protein 5 ( NS5) is a key activity for viral RNA replication. In this study, crystal structures of enzymatically active and inactive WNV RdRp domains were determined at 3.0- and 2.35-angstrom resolution, respectively. The determined structures were shown to be mostly similar to the RdRps of the Flaviviridae members hepatitis C and bovine viral diarrhea virus, although with unique elements characteristic for the WNVRdRp. Using a reverse genetic system, residues involved in putative interactions between the RNA-cap methyltransferase ( MTase) and the RdRp domain of Flavivirus NS5 were identified. This allowed us to propose a model for the structure of the full-length WNV NS5 by in silico docking of the WNV MTase domain ( modeled from our previously determined structure of the DENV MTase domain) onto the RdRp domain. The Flavivirus RdRp domain structure determined here should facilitate both the design of anti-Flavivirus drugs and structure-function studies of the Flavivirus replication complex in which the multifunctional NS5 protein plays a central role

Methionine Sulfoxide Reductases Are Essential for Virulence of Salmonella Typhimurium

Production of reactive oxygen species represents a fundamental innate defense against microbes in a diversity of host organisms. Oxidative stress, amongst others, converts peptidyl and free methionine to a mixture of methionine-S- (Met-S-SO) and methionine-R-sulfoxides (Met-R-SO). To cope with such oxidative damage, methionine sulfoxide reductases MsrA and MsrB are known to reduce MetSOs, the former being specific for the S-form and the latter being specific for the R-form. However, at present the role of methionine sulfoxide reductases in the pathogenesis of intracellular bacterial pathogens has not been fully detailed. Here we show that deletion of msrA in the facultative intracellular pathogen Salmonella (S.) enterica serovar Typhimurium increased susceptibility to exogenous H2O2, and reduced bacterial replication inside activated macrophages, and in mice. In contrast, a ΔmsrB mutant showed the wild type phenotype. Recombinant MsrA was active against free and peptidyl Met-S-SO, whereas recombinant MsrB was only weakly active and specific for peptidyl Met-R-SO. This raised the question of whether an additional Met-R-SO reductase could play a role in the oxidative stress response of S. Typhimurium. MsrC is a methionine sulfoxide reductase previously shown to be specific for free Met-R-SO in Escherichia (E.) coli. We tested a ΔmsrC single mutant and a ΔmsrBΔmsrC double mutant under various stress conditions, and found that MsrC is essential for survival of S. Typhimurium following exposure to H2O2, as well as for growth in macrophages, and in mice. Hence, this study demonstrates that all three methionine sulfoxide reductases, MsrA, MsrB and MsrC, facilitate growth of a canonical intracellular pathogen during infection. Interestingly MsrC is specific for the repair of free methionine sulfoxide, pointing to an important role of this pathway in the oxidative stress response of Salmonella Typhimurium

Fidelity Variants of RNA Dependent RNA Polymerases Uncover an Indirect, Mutagenic Activity of Amiloride Compounds

In a screen for RNA mutagen resistance, we isolated a high fidelity RNA dependent RNA polymerase (RdRp) variant of Coxsackie virus B3 (CVB3). Curiously, this variant A372V is also resistant to amiloride. We hypothesize that amiloride has a previously undescribed mutagenic activity. Indeed, amiloride compounds increase the mutation frequencies of CVB3 and poliovirus and high fidelity variants of both viruses are more resistant to this effect. We hypothesize that this mutagenic activity is mediated through alterations in intracellular ions such as Mg2+ and Mn2+, which in turn increase virus mutation frequency by affecting RdRp fidelity. Furthermore, we show that another amiloride-resistant RdRp variant, S299T, is completely resistant to this mutagenic activity and unaffected by changes in ion concentrations. We show that RdRp variants resist the mutagenic activity of amiloride via two different mechanisms: 1) increased fidelity that generates virus populations presenting lower basal mutation frequencies or 2) resisting changes in divalent cation concentrations that affect polymerase fidelity. Our results uncover a new antiviral approach based on mutagenesis

The X-Ray Crystal Structure of Escherichia coli Succinic Semialdehyde Dehydrogenase; Structural Insights into NADP+/Enzyme Interactions

In mammals succinic semialdehyde dehydrogenase (SSADH) plays an essential role in the metabolism of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) to succinic acid (SA). Deficiency of SSADH in humans results in elevated levels of GABA and gamma-Hydroxybutyric acid (GHB), which leads to psychomotor retardation, muscular hypotonia, non-progressive ataxia and seizures. In Escherichia coli, two genetically distinct forms of SSADHs had been described that are essential for preventing accumulation of toxic levels of succinic semialdehyde (SSA) in cells.Here we structurally characterise SSADH encoded by the E coli gabD gene by X-ray crystallographic studies and compare these data with the structure of human SSADH. In the E. coli SSADH structure, electron density for the complete NADP+ cofactor in the binding sites is clearly evident; these data in particular revealing how the nicotinamide ring of the cofactor is positioned in each active site.Our structural data suggest that a deletion of three amino acids in E. coli SSADH permits this enzyme to use NADP+, whereas in contrast the human enzyme utilises NAD+. Furthermore, the structure of E. coli SSADH gives additional insight into human mutations that result in disease

Deep Sequencing Whole Transcriptome Exploration of the σE Regulon in Neisseria meningitidis

Bacteria live in an ever-changing environment and must alter protein expression promptly to adapt to these changes and survive. Specific response genes that are regulated by a subset of alternative σ70-like transcription factors have evolved in order to respond to this changing environment. Recently, we have described the existence of a σE regulon including the anti-σ-factor MseR in the obligate human bacterial pathogen Neisseria meningitidis. To unravel the complete σE regulon in N. meningitidis, we sequenced total RNA transcriptional content of wild type meningococci and compared it with that of mseR mutant cells (ΔmseR) in which σE is highly expressed. Eleven coding genes and one non-coding gene were found to be differentially expressed between H44/76 wildtype and H44/76ΔmseR cells. Five of the 6 genes of the σE operon, msrA/msrB, and the gene encoding a pepSY-associated TM helix family protein showed enhanced transcription, whilst aniA encoding a nitrite reductase and nspA encoding the vaccine candidate Neisserial surface protein A showed decreased transcription. Analysis of differential expression in IGRs showed enhanced transcription of a non-coding RNA molecule, identifying a σE dependent small non-coding RNA. Together this constitutes the first complete exploration of an alternative σ-factor regulon in N. meningitidis. The results direct to a relatively small regulon indicative for a strictly defined response consistent with a relatively stable niche, the human throat, where N. meningitidis resides

Engineering an aldehyde dehydrogenase toward its substrates, 3-hydroxypropanal and NAD(+), for enhancing the production of 3-hydroxypropionic acid

3-Hydroxypropionic acid (3-HP) can be produced via the biological route involving two enzymatic reactions: dehydration of glycerol to 3-hydroxypropanal (3-HPA) and then oxidation to 3-HP. However, commercial production of 3-HP using recombinant microorganisms has been hampered with several problems, some of which are associated with the toxicity of 3-HPA and the efficiency of NAD(+) regeneration. We engineered a-ketoglutaric semialdehyde dehydrogenase (KGSADH) from Azospirillum brasilense for the second reaction to address these issues. The residues in the binding sites for the substrates, 3-HPA and NAD(+), were randomized, and the resulting libraries were screened for higher activity. Isolated KGSADH variants had significantly lower Km values for both the substrates. The enzymes also showed higher substrate specificities for aldehyde and NAD(+), less inhibition by NADH, and greater resistance to inactivation by 3-HPA than the wild-type enzyme. A recombinant Pseudomonas denitrificans strain with one of the engineered KGSADH variants exhibited less accumulation of 3-HPA, decreased levels of inactivation of the enzymes, and higher cell growth than that with the wild-type KGSADH. The flask culture of the P. denitrificans strain with the mutant KGSADH resulted in about 40% increase of 3-HP titer (53 mM) compared with that using the wild-type enzyme (37 mM)

Modular Oxime Formation by a trans-AT Polyketide Synthase

Modular trans-acyltransferase polyketide synthases (trans-AT PKSs) are enzymatic assembly lines that biosynthesize complex polyketide natural products. Relative to their better studied cis-AT counterparts, the trans-AT PKSs introduce remarkable chemical diversity into their polyketide products. A notable example is the lobatamide A PKS, which incorporates a methylated oxime. Here we demonstrate biochemically that this functionality is installed on-line by an unusual oxygenase-containing bimodule. Furthermore, analysis of the oxygenase crystal structure coupled with site-directed mutagenesis allows us to propose a model for catalysis, as well as identifying key protein-protein interactions that support this chemistry. Overall, our work adds oxime-forming machinery to the biomolecular toolbox available for trans-AT PKS engineering, opening the way to introducing such masked aldehyde functionalities into diverse polyketides.ISSN:1433-7851ISSN:1521-3773ISSN:0570-083

The Chlamydomonas reinhardtii Molybdenum Cofactor Enzyme crARC Has a Zn-Dependent Activity and Protein Partners Similar to Those of Its Human Homologue ▿

The ARC (amidoxime reducing component) proteins are molybdenum cofactor (Moco) enzymes named hmARC1 and hmARC2 (human ARCs [hmARCs]) in humans and YcbX in Escherichia coli. They catalyze the reduction of a broad range of N-hydroxylated compounds (NHC) using reducing power supplied by other proteins. Some NHC are prodrugs or toxic compounds. YcbX contains a ferredoxin (Fd) domain and requires the NADPH flavin reductase CysJ to reduce NHC. In contrast, hmARCs lack the Fd domain and require a human cytochrome b5 (hCyt b5) and a human NADH Cyt b5 reductase (hCyt b5-R) to reduce NHC. The ARC proteins in the plant kingdom are uncharacterized. We demonstrate that Chlamydomonas reinhardtii mutants defective in Moco biosynthesis genes are sensitive to the NHC N6-hydroxylaminopurine (HAP). The Chlamydomonas reinhardtii ARC protein crARC has been purified and characterized. The six Chlamydomonas Fds were isolated, but none of them are required by crARC to reduce HAP. We have also purified and characterized five C. reinhardtii Cyt b5 (crCyt b5) and two flavin reductases, one that is NADPH dependent (crCysJ) and one that is NADH dependent (crCyt b5-R). The data show that crARC uses crCyt b5-1 and crCyt b5-R to reduce HAP. The crARC has a Zn-dependent activity, and the presence of Zn increases its Vmax more than 14-fold. In addition, all five cysteines of crARC were substituted by alanine, and we demonstrate that the fully conserved cysteine 252 is essential for both Moco binding and catalysis. Therefore, it is proposed that crARC belongs to the sulfite oxidase family of Moco enzymes

Amiloride Is a Competitive Inhibitor of Coxsackievirus B3 RNA Polymerase ▿

Amiloride and its derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were previously shown to inhibit coxsackievirus B3 (CVB3) RNA replication in cell culture, with two amino acid substitutions in the viral RNA-dependent RNA polymerase 3Dpol conferring partial resistance of CVB3 to these compounds (D. N. Harrison, E. V. Gazina, D. F. Purcell, D. A. Anderson, and S. Petrou, J. Virol. 82:1465–1473, 2008). Here we demonstrate that amiloride and EIPA inhibit the enzymatic activity of CVB3 3Dpol in vitro, affecting both VPg uridylylation and RNA elongation. Examination of the mechanism of inhibition of 3Dpol by amiloride showed that the compound acts as a competitive inhibitor, competing with incoming nucleoside triphosphates (NTPs) and Mg2+. Docking analysis suggested a binding site for amiloride and EIPA in 3Dpol, located in close proximity to one of the Mg2+ ions and overlapping the nucleotide binding site, thus explaining the observed competition. This is the first report of a molecular mechanism of action of nonnucleoside inhibitors against a picornaviral RNA-dependent RNA polymerase