The isolation of L(+)-methionine sulphoxide from the blowfly Phormia regina Meigen.
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ABSTRACT:
SUBMITTER: Lucas F
PROVIDER: S-EPMC1265158 | biostudies-other | 1966 Aug
REPOSITORIES: biostudies-other
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Project description:Methionine sulphoxide reductase maintains adhesin function during oxidative stress. Using Streptococcus gordonii as a model, we now show the mechanistic basis of adhesin maintenance provided by MsrA. In biofilms, S. gordonii selectively expresses the msrA gene. When the wild-type strain was grown with exogenous hydrogen peroxide (H(2)O(2)), msrA-specific mRNA expression significantly increased, while acid production was unaffected. In the presence of H(2)O(2), a msrA-deletion mutant (?MsrA) showed a 6 h delay in lag phase growth, a 30% lower yield of H(2)O(2), significantly greater inhibition by H(2)O(2) on agar plates (reversed by complementation), 30% less adhesion to saliva-coated hydroxyapatite, 87% less biofilm formation and an altered electrophoretic pattern of SspAB protein adhesins. Using mass spectrometry, methionine residues in the Met-rich central region of SspB were shown to be oxidized by H(2)O(2) and reduced by MsrA. In intact wild-type cells, MsrA colocalized with a cell wall-staining dye, and MsrA was detected in both cell wall and cytosolic fractions. To maintain normal adhesion and biofilm function of S. gordonii in response to exogenous oxidants therefore msrA is upregulated, methionine oxidation of adhesins and perhaps other proteins is reversed, and adhesion and biofilm formation is maintained.
| S-EPMC3098138 | biostudies-literature
Project description:Methionine residues and iron-sulphur (FeS) clusters are primary targets of reactive oxygen species in the proteins of micro-organisms. Here, we show that methionine redox modifications help to preserve essential FeS cluster activities in yeast. Mutants defective for the highly conserved methionine sulphoxide reductases (MSRs; which re-reduce oxidized methionines) are sensitive to many pro-oxidants, but here exhibited an unexpected copper resistance. This phenotype was mimicked by methionine sulphoxide supplementation. Microarray analyses highlighted several Cu and Fe homeostasis genes that were upregulated in the mxrDelta double mutant, which lacks both of the yeast MSRs. Of the upregulated genes, the Cu-binding Fe transporter Fet3p proved to be required for the Cu-resistance phenotype. FET3 is known to be regulated by the Aft1 transcription factor, which responds to low mitochondrial FeS-cluster status. Here, constitutive Aft1p expression in the wild-type reproduced the Cu-resistance phenotype, and FeS-cluster functions were found to be defective in the mxrDelta mutant. Genetic perturbation of FeS activity also mimicked FET3-dependent Cu resistance. 55Fe-labelling studies showed that FeS clusters are turned over more rapidly in the mxrDelta mutant than the wild-type, consistent with elevated oxidative targeting of the clusters in MSR-deficient cells. The potential underlying molecular mechanisms of this targeting are discussed. Moreover, the results indicate an important new role for cellular MSR enzymes in helping to protect the essential function of FeS clusters in aerobic settings.
| S-EPMC2653451 | biostudies-literature
Project description:A cytochrome c haem ligand, methionine-80, was photo-oxidized to methionine sulphoxide and the subsequent changes in redox properties and ligand binding were monitored kinetically. Isoelectric focusing of the product showed the presence of a single oxidized species, capable of binding CO when reduced. The binding of CO to the reduced protein was followed in stopped-flow experiments, which revealed the presence of two binding processes, at neutral pH, with rate constants of K+1 = 3.4 X 10(3)M-1-S-1 and k+2 = 5.80 X 10(2)M-1-S-1. When CO was photolytically dissociated from the reduced protein two recombination processes were observed with rates almost identical with those observed in the stopped-flow experiments (k+1 = 3.3 X 10(3)M-1-S-1 and k+2 = 6.0 X 10(2)M-1-S-1). These findings provide evidence of two reduced forms of the protein. The reduction of [methionine sulphoxide]cytochrome c by Cr2+ at neutral pH in stopped-flow experiments showed the presence of a single second-order reduction process (k = 7.2 X 10(3)M-1-S-1, activation energy = 44kJ/mol) and one first-order process. This protein was compared with some other chemically modified cytochromes.
| S-EPMC1164161 | biostudies-other
Project description:MsrA (methionine sulphoxide reductase A) is an antioxidant repair enzyme that reduces oxidized methionine to methionine. Moreover, the oxidation of methionine residues in proteins is considered to be an important consequence of oxidative damage to cells. To understand mechanisms of human msrA gene expression and regulation, we cloned and characterized the 5' promoter region of the human msrA gene. Using 5'-RACE (rapid amplification of cDNA ends) analysis of purified mRNA from human cells, we located the transcription initiation site 59 nt upstream of the reference MsrA mRNA sequence, GenBank accession number BC 054033. The 1.3 kb of sequence located upstream of the first exon of msrA gene was placed upstream of the luciferase reporter gene in a pGL3-Basic vector and transfected into different cell lines. Sequentially smaller fragments of the msrA promoter region were generated by PCR, and expression levels were monitored from these constructs within HEK-293 and MCF7 human cell lines. Analysis of deletion constructs revealed differences in promoter activity in these cell lines. In HEK-293 cells, the promoter activity was constant from the minimal promoter region to the longest fragment obtained. On the other hand, in MCF7 cells we detected a down-regulation in the longest fragment. Mutation of a putative negative regulatory region that is located between -209 and -212 bp (the CCAA box) restored promoter activity in MCF7 cells. The location of the msrA promoter will facilitate analysis of the transcriptional regulation of this gene in a variety of pathological contexts.
| S-EPMC1383691 | biostudies-literature
Project description:Methionine sulphoxide reductases (Msr) reduce methionine sulphoxide to methionine and protect bacteria against reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Many organisms express both MsrA, active against methionine-(S)-sulphoxide, and MsrB, active against methionine-(R)-sulphoxide. Mycobacterium tuberculosis (Mtb) expresses MsrA, which protects DeltamsrA-Escherichia coli from ROI and RNI. However, the function of MsrA in Mtb has not been defined, and it is unknown whether Mtb expresses MsrB. We identified MsrB as the protein encoded by Rv2674 in Mtb and confirmed the distinct stereospecificities of recombinant Mtb MsrA and MsrB. We generated strains of Mtb deficient in MsrA, MsrB or both and complemented the mutants. Lysates of singly deficient strains displayed half as much Msr activity as wild type against N-acetyl methionine sulphoxide. However, in contrast to other bacteria, single mutants were no more vulnerable than wild type to killing by ROI/RNI. Only Mtb lacking both MsrA and MsrB was more readily killed by nitrite or hypochlorite. Thus, MsrA and MsrB contribute to the enzymatic defences of Mtb against ROI and RNI.
| S-EPMC3697012 | biostudies-literature
Project description:AimsThe enzyme catechol-O-methyltransferase (COMT) plays a primary role in the metabolism of catecholamine neurotransmitters and is implicated in the modulation of cognitive and emotional responses. The best characterized single nucleotide polymorphism (SNP) of the COMT gene consists of a valine (Val)-to-methionine (Met) substitution at codon 108/158. The Met-containing variant confers a marked reduction in COMT catalytic activity. We recently showed that the activity of recombinant COMT is positively regulated by the enzyme Met sulphoxide reductase (MSR), which counters the oxidation of Met residues of proteins. The current study was designed to assess whether brain COMT activity may be correlated to MSR in an allele-dependent fashion.MethodsCOMT and MSR activities were measured from post-mortem samples of prefrontal cortices, striata and cerebella of 32 subjects by using catechol and dabsyl-Met sulphoxide as substrates, respectively. Allelic discrimination of COMT Val(108/185) Met SNP was performed using the Taqman 5'nuclease assay.ResultsOur studies revealed that, in homozygous carriers of Met, but not Val alleles, the activity of COMT and MSR was significantly correlated throughout all tested brain regions.ConclusionThese results suggest that the reduced enzymatic activity of Met-containing COMT may be secondary to Met sulphoxidation and point to MSR as a key molecular determinant for the modulation of COMT activity.
| S-EPMC4500745 | biostudies-literature
Project description:Peptide methionine sulphoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous enzyme catalysing the reduction of methionine sulphoxide to methionine in proteins, while the glutathione S-transferases (GSTs) are a major family of detoxification enzymes. A gene homologous to MsrA was identified in a chromosomal fragment from the bacterium Ochrobactrum anthropi, and this gene is located just downstream of a GST gene identified previously (OaGST) [Favaloro, Tamburro, Angelucci, De Luca, Melino, Di Ilio and Rotilio (1998) Biochem. J. 335, 573-579]. This raises the question of whether the products of these two genes may be involved in a common cellular protection function. To test this hypothesis, the hypothetical MsrA protein has been overexpressed in Escherichia coli as a functional 51 kDa GST fusion protein. Following cleavage with thrombin and purification, the soluble 24 kDa protein showed MsrA activity with N-acetylmethionine sulphoxide as substrate, as well as with other sulphoxide compounds. Therefore polyclonal antibodies were raised against the recombinant protein, and the modulation of MsrA in this bacterium, grown in the presence of different stimulants simulating several stress conditions, was investigated. The level of expression of MsrA was detected both by measuring the mRNA level and by immunoblotting experiments, in addition to measuring its catalytic activity. MsrA is a constitutive enzyme which is also inducible by chemical stress involving phenolic compounds such as phenol and 4-chlorophenol. Recently we reported that the GST of this bacterium, like MsrA, is only modulated by toxic chemical compounds [Favaloro, Tamburro, Trofino, Bologna, Rotilio and Heipieper (2000) Biochem. J. 346, 553-559]; therefore this is the first indication of a co-induction of the MsrA and GST enzymes during chemical stress.
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Project description:Proteins are sensitive to reactive oxygen species, and the accumulation of oxidized proteins has been implicated in the aging process and in other age-related pathologies. In proteins, methionine residues are especially sensitive to oxidation, leading to S - and R -methionine sulphoxide diastereoisomers, the reversion of which is achieved by the peptide methionine sulphoxide reductases MsrA and MsrB respectively. The MsrA enzyme, in addition to its role in repair, forms part of the reactive oxygen species scavenging systems that are important in cellular antioxidant defence. MsrA is present in most living organisms, and the mammalian enzyme has been detected in all tissues investigated. In the present study, we investigated the subcellular distribution of MsrA in rat liver cells. Since it seemed likely that MsrA may be localized in areas where reactive oxygen species are produced, rat liver mitochondrial matrix and cytosolic extracts were prepared. The presence of MsrA was assayed in these subcellular compartments by monitoring peptide methionine sulphoxide reductase enzymic activity, by Western blotting and by in situ immunolocalization by electron microscopy using a specific antibody. Moreover, MsrA was identified by MS in a partially purified cytosolic fraction and in a mitochondrial matrix crude extract. Rat MsrA isoforms are encoded by a single gene, and it is suggested that the precursor of the mitochondrial form contains an N-terminal cleavable signal sequence that localizes the MsrA to this organelle. Finally, two-dimensional gel electrophoresis followed by Western-blot analysis of partially purified MsrA from the cytosol and mitochondria, and comparison with the two-dimensional patterns of oxidized recombinant MsrA, revealed oxidative modifications of cysteine residues.
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Project description:Human immunodeficiency viruses encode a homodimeric protease that is essential for the production of infectious virus. Previous studies have shown that HIV-1 protease is susceptible to oxidative inactivation at the dimer interface at Cys-95, a process that can be reversed both chemically and enzymically. Here we demonstrate a related yet distinct mechanism of reversible inactivation of the HIV-2 protease. Exposure of the HIV-2 protease to H(2)O(2) resulted in conversion of the two methionine residues (Met-76 and Met-95) to methionine sulphoxide as determined by amino acid analysis and mass spectrometry. This oxidation completely inactivated protease activity. However, the activity could be restored (up to 40%) after exposure of the oxidized protease to methionine sulphoxide reductase. This treatment resulted in the reduction of methionine sulphoxide 95 but not methionine sulphoxide 76 to methionine, as determined by peptide mapping/mass spectrometry. We also found that exposure of immature HIV-2 particles to H(2)O(2) led to the inhibition of polyprotein processing in maturing virus particles comparable to that demonstrated for HIV-1 particles. Thus oxidative inactivation of the HIV protease in vitro and in maturing viral particles is not restricted to the type 1 proteases. These studies indicate that two distinct retroviral proteases are susceptible to inactivation after a very minor modification at residue 95 of the dimer interface and suggest that the dimer interface might be a viable target for the development of novel protease inhibitors.
| S-EPMC1220854 | biostudies-other
Project description:1. The isolation of an o-diphenol oxidase from an acetone-dried powder of late-third-instar larvae of Calliphora erythrocephala was investigated. An insoluble and micro-crystalline fraction containing the enzyme activity was obtained after fractionating extracts of the acetone-dried powder with (NH4)2SO4 and acetone. 2. This fraction can be solubilized in 0.1% sodium dodecyl sulphate without loss of activity. 3. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate shows that the o-diphenol oxidase is a minor component of the extracts from the acetone-dried powder. 4. The o-diphenol oxidase was purified by zonal centrifugation on a sucrose density gradient in the presence of sodium dodecyl sulphate. 5. The amino acid composition of the purified enzyme resembles that of some other o-diphenol oxidases. 6. The subunit composition of the o-diphenol oxidase is discussed.
| S-EPMC1165677 | biostudies-other