Dataset Information

Synergistic roles of Helicobacter pylori methionine sulfoxide reductase and GroEL in repairing oxidant-damaged catalase.

ABSTRACT: Hypochlorous acid (HOCl) produced via the enzyme myeloperoxidase is a major antibacterial oxidant produced by neutrophils, and Met residues are considered primary amino acid targets of HOCl damage via conversion to Met sulfoxide. Met sulfoxide can be repaired back to Met by methionine sulfoxide reductase (Msr). Catalase is an important antioxidant enzyme; we show it constitutes 4-5% of the total Helicobacter pylori protein levels. msr and katA strains were about 14- and 4-fold, respectively, more susceptible than the parent to killing by the neutrophil cell line HL-60 cells. Catalase activity of an msr strain was much more reduced by HOCl exposure than for the parental strain. Treatment of pure catalase with HOCl caused oxidation of specific MS-identified Met residues, as well as structural changes and activity loss depending on the oxidant dose. Treatment of catalase with HOCl at a level to limit structural perturbation (at a catalase/HOCl molar ratio of 1:60) resulted in oxidation of six identified Met residues. Msr repaired these residues in an in vitro reconstituted system, but no enzyme activity could be recovered. However, addition of GroEL to the Msr repair mixture significantly enhanced catalase activity recovery. Neutrophils produce large amounts of HOCl at inflammation sites, and bacterial catalase may be a prime target of the host inflammatory response; at high concentrations of HOCl (1:100), we observed loss of catalase secondary structure, oligomerization, and carbonylation. The same HOCl-sensitive Met residue oxidation targets in catalase were detected using chloramine-T as a milder oxidant.

SUBMITTER: Mahawar M

PROVIDER: S-EPMC3099729 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Synergistic roles of Helicobacter pylori methionine sulfoxide reductase and GroEL in repairing oxidant-damaged catalase.

Mahawar Manish M Tran ViLinh V Sharp Joshua S JS Maier Robert J RJ

The Journal of biological chemistry 20110401 21

Hypochlorous acid (HOCl) produced via the enzyme myeloperoxidase is a major antibacterial oxidant produced by neutrophils, and Met residues are considered primary amino acid targets of HOCl damage via conversion to Met sulfoxide. Met sulfoxide can be repaired back to Met by methionine sulfoxide reductase (Msr). Catalase is an important antioxidant enzyme; we show it constitutes 4-5% of the total Helicobacter pylori protein levels. msr and katA strains were about 14- and 4-fold, respectively, mor ...[more]

PMID: 21460217

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Project description:Methionine residues in proteins are susceptible to oxidation by reactive oxygen species, but can be repaired via reduction of the resulting methionine sulfoxides by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). However, the identity of all methionine sulfoxide reductases involved, their cellular locations and relative contributions to the overall pathway are poorly understood. Here, we describe a methionine-R-sulfoxide reduction system in mammals, in which two MsrB homologues were previously described. We found that human and mouse genomes possess three MsrB genes and characterized their protein products, designated MsrB1, MsrB2, and MsrB3. MsrB1 (Selenoprotein R) was present in the cytosol and nucleus and exhibited the highest methionine-R-sulfoxide reductase activity because of the presence of selenocysteine (Sec) in its active site. Other mammalian MsrBs contained cysteine in place of Sec and were less catalytically efficient. MsrB2 (CBS-1) resided in mitochondria. It had high affinity for methionine-R-sulfoxide, but was inhibited by higher concentrations of the substrate. The human MsrB3 gene gave rise to two protein forms, MsrB3A and MsrB3B. These were generated by alternative splicing that introduced contrasting N-terminal and C-terminal signals, such that MsrB3A was targeted to the endoplasmic reticulum and MsrB3B to mitochondria. We found that only mitochondrial forms of mammalian MsrBs (MsrB2 and MsrB3B) could compensate for MsrA and MsrB deficiency in yeast. All mammalian MsrBs belonged to a group of zinc-containing proteins. The multiplicity of MsrBs contrasted with the presence of a single mammalian MsrA gene as well as with the occurrence of single MsrA and MsrB genes in yeast, fruit flies, and nematodes. The data suggested that different cellular compartments in mammals maintain a system for repair of oxidized methionine residues and that this function is tuned in enzyme- and stereo-specific manner.

Project description:The N-terminal sequence of a protein, originally described as an adhesin of Helicobacter pylori, was used in an oligonucleotide-based screening procedure of an H. pylori plasmid library in Escherichia coli. Five independent plasmid clones were isolated, all mapping to the same chromosomal region and encoding the H. pylori catalase. The gene, designated katA, comprises 1,518 nucleotides and encodes a putative protein of 505 amino acids with a predicted Mr of 58,599. A second open reading frame, orf2, encoding a putative 32,715-Da protein of unknown function, follows katA. The transcriptional start site of katA mRNA was determined, but no typical consensus promoter sequence was present. A potential binding site for the Fur protein is located upstream of katA. When introduced into the catalase-deficient E. coli double-mutant UM255, the cloned gene readily complemented E. coli for catalase activity. H. pylori KatA is highly homologous to catalases in both prokaryotes and eukaryotes, with the highest homology being shown to Bordetella pertussis (64.9%), Bacteroides fragilis (59.8%), and Haemophilus influenzae (57.9%) catalases. Transposon insertion mutants were generated in three independent H. pylori strains by TnMax5-mediated transposon shuttle mutagenesis. In contrast to the wild-type strains, no significant catalase-specific enzymatic activity could be detected in the mutant strains, consistent with the fact that no additional katA-homologous gene copies were found in the H. pylori chromosome. No significant difference between wild-type and mutant strains for binding to epithelial cells was apparent, suggesting that KatA is not involved in H. pylori adhesion. The cloning and genetic characterization of katA are essential steps for further investigation of the role of catalase in the defense of H. pylori against oxygen-dependent killing mechanisms by polymorphonuclear granulocytes, a process not well understood for this chronically persisting pathogen.

Dataset Information

Synergistic roles of Helicobacter pylori methionine sulfoxide reductase and GroEL in repairing oxidant-damaged catalase.

Publications

Synergistic roles of Helicobacter pylori methionine sulfoxide reductase and GroEL in repairing oxidant-damaged catalase.

Similar Datasets

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