Activity, tissue distribution and site-directed mutagenesis of a human peptide methionine sulfoxide reductase of type B: hCBS1

Staphylococcus aureus produces 3 MsrA enzymes (MsrA1, MsrA2, and MsrA3) and 1 MsrB enzyme. The genes encoding MsrA1 and MsrB are the first and second genes of a 4-gene operon in S. aureus. In a previous study, MsrA1-deficient S. aureus cells showed increased sensitivity to oxidative stress conditions in spite of a higher production of MsrB. In this study, an msrB mutant of S. aureus was created by site-directed mutagenesis that left the first gene of this locus, msrA1, intact. Studies with this mutant suggest that a deletion of MsrB increases resistance of S. aureus to H2O2 and oxacillin and that the mutant cells produce a higher level of carotenoids relative to wild-type S. aureus cells.

Download Full-text

Ribose 5-phosphate isomerase type B from Trypanosoma cruzi: kinetic properties and site-directed mutagenesis reveal information about the reaction mechanism

Biochemical Journal ◽

10.1042/bj20061049 ◽

2006 ◽

Vol 401 (1) ◽

pp. 279-285 ◽

Cited By ~ 30

Author(s):

Ana L. Stern ◽

Emmanuel Burgos ◽

Laurent Salmon ◽

Juan J. Cazzulo

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Pentose Phosphate ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Kinetic Properties ◽

Type B ◽

Nucleotide Synthesis ◽

Gene Coding ◽

Genes Encoding

Trypanosoma cruzi, the human parasite that causes Chagas disease, contains a functional pentose phosphate pathway, probably essential for protection against oxidative stress and also for R5P (ribose 5-phosphate) production for nucleotide synthesis. The haploid genome of the CL Brener clone of the parasite contains one gene coding for a Type B Rpi (ribose 5-phosphate isomerase), but genes encoding Type A Rpis, most frequent in eukaryotes, seem to be absent. The RpiB enzyme was expressed in Escherichia coli as a poly-His tagged active dimeric protein, which catalyses the reversible isomerization of R5P to Ru5P (ribulose 5-phos-phate) with Km values of 4 mM (R5P) and 1.4 mM (Ru5P).4-Phospho-D-erythronohydroxamic acid, an analogue to the reaction intermediate when the Rpi acts via a mechanism involving the formation of a 1,2-cis-enediol, inhibited the enzyme competi-tively, with an IC50 value of 0.7 mM and a Ki of 1.2 mM. Site-directed mutagenesis allowed the demonstration of a role for His102, but not for His138, in the opening of the ribose furanosic ring. A major role in catalysis was confirmed for Cys69, since the C69A mutant was inactive in both forward and reverse directions of the reaction. The present paper contributes to the know-ledge of the mechanism of the Rpi reaction; in addition, the absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for chemotherapy of Chagas disease.

Download Full-text

Staphylococcus aureus peptide methionine sulfoxide reductases protect from human whole blood killing.

Infection and Immunity ◽

10.1128/iai.00146-21 ◽

2021 ◽

Author(s):

William N. Beavers ◽

Ashley L. DuMont ◽

Andrew J. Monteith ◽

K. Nichole Maloney ◽

Keri A. Tallman ◽

...

Keyword(s):

Oxidative Stress ◽

Staphylococcus Aureus ◽

Protein Oxidation ◽

Whole Blood ◽

Methionine Sulfoxide ◽

Methionine Sulfoxide Reductase ◽

Human Whole Blood ◽

Infection Models ◽

Intraperitoneal Infection ◽

Peptide Methionine Sulfoxide Reductase

The generation of oxidative stress is a host strategy used to control Staphylococcus aureus infections. Sulfur containing amino acids, cysteine and methionine, are particularly susceptible to oxidation because of the inherent reactivity of sulfur. Due to the constant threat of protein oxidation, many systems evolved to protect S. aureus from protein oxidation or to repair protein oxidation after it occurs. The S. aureus peptide methionine sulfoxide reductase (Msr) system reduces methionine sulfoxide to methionine. Staphylococci have four Msr enzymes, which all perform this reaction. Deleting all four msr genes in USA300 LAC (Δmsr) sensitizes S. aureus to hypochlorous acid (HOCl) killing, however, Δmsr does not exhibit increased sensitivity to H2O2 stress or superoxide anion stress generated by paraquat or pyocyanin. Consistent with increased susceptibility to HOCl killing, Δmsr is slower to recover following co-culture with both murine and human neutrophils than USA300 wildtype. Δmsr is attenuated for dissemination to the spleen following murine intraperitoneal infection and exhibits reduced bacterial burdens in a murine skin infection model. Notably, no differences in bacterial burdens were observed in any organ following murine intravenous infection. Consistent with these observations, USA300 wildtype and Δmsr have similar survival phenotypes when incubated with murine whole blood. However, Δmsr is killed more efficiently by human whole blood. These findings indicate that species-specific immune cell composition of the blood may influence the importance of Msr enzymes during S. aureus infection of the human host. IMPORTANCE Oxidative stress is a host defense strategy to control bacterial infections, and bacteria have evolved systems to counteract this innate immune defense. Here we investigate the peptide methionine sulfoxide reductase system in Staphylococcus aureus that repairs oxidized methionine residues in proteins, preventing the need to resynthesize damaged proteins de novo. Most organisms have an Msr system, and in S. aureus these enzymes are protective against HOCl killing, the major oxidant produced by neutrophils. The S. aureus Msr system does not have a significant contribution to pathogenesis in bacteremia murine infection models but does protect S. aureus in both skin and intraperitoneal infection models. Strains lacking Msr activity are killed equivalently to wildtype by murine whole blood, and Δmsr is more sensitive to killing by human whole blood than the wildtype strain. These data identify the Msr enzymes as important and potentially specific factors for S. aureus pathogenesis in the human host.

Download Full-text