Reduction of Sulindac to its active metabolite, sulindac sulfide: assay and role of the methionine sulfoxide reductase system.

Sulindac is a known anti-inflammatory drug that functions by inhibition of cyclooxygenases 1 and 2 (COX). There has been recent interest in Sulindac and other non-steroidal anti-inflammatory drugs (NSAID) because of their anti-tumor activity against colorectal cancer. Studies with sulindac have indicated that it may also function as an anti-tumor agent by stimulating apoptosis. Sulindac is a pro-drug, containing a methyl sulfoxide group, that must be reduced to sulindac sulfide to be active as a COX inhibitor. In the present studies we have developed a simple assay to measure sulindac reduction and tested sulindac as a substrate for 6 known members of the methionine sulfoxide reductase (Msr) family that have been identified in Escherichia coli. Only MsrA and a membrane associated Msr can reduce sulindac to the active sulfide. The reduction of sulindac also has been demonstrated in extracts of calf liver, kidney, and brain. Sulindac reductase activity is also present in mitochondria and microsomes.

New membrane-associated and soluble peptide methionine sulfoxide reductases in Escherichia coli.

It is known that reactive oxygen species can oxidize methionine residues in proteins in a non-stereospecific manner, and cells have mechanisms to reverse this damage. MsrA and MsrB are members of the methionine sulfoxide family of enzymes that specifically reduce the S and R forms, respectively, of methionine sulfoxide in proteins. However, in Escherichia coli the level of MsrB activity is very low which suggested that there may be other enzymes capable of reducing the R epimer of methionine sulfoxide in proteins. Employing a msrA/B double mutant, a new peptide methionine sulfoxide reductase activity has been found associated with membrane vesicles from E. coli. Both the R and S forms of N-acetylmethionine sulfoxide, D-ala-met(o)-enkephalin and methionine sulfoxide, are reduced by this membrane associated activity. The reaction requires NADPH and may explain, in part, how the R form of methionine sulfoxide in proteins is reduced in E. coli. In addition, a new soluble Msr activity was also detected in the soluble extracts of the double mutant that specifically reduces the S epimer of met(o) in proteins.

A methionine sulfoxide reductase in Escherichia coli that reduces the R enantiomer of methionine sulfoxide.

It is known that Escherichia coli methionine mutants can grow on both enantiomers of methionine sulfoxide (met(o)), i.e., met-R-(o) or met-S-(o), indicating the presence of enzymes in E. coli that can reduce each of these enantiomers to methionine (met). Previous studies have identified two members of the methionine sulfoxide reductase (Msr) family of enzymes, MsrA and fSMsr, that could reduce free met-S-(o), but the reduction of free met-R-(o) to met has not been elucidated. One possible candidate is MsrB which is known to reduce met-R-(o) in proteins to met. However, free met-R-(o) is a very poor substrate for MsrB and the level of MsrB activity in E. coli extracts is very low. A new member of the Msr family (fRMsr) has been identified in E. coli extracts that reduces free met-R-(o) to met. Partial purification of FRMsr has been obtained using extracts from an MsrA/MsrB double mutant of E. coli.

The outer membrane localization of the Neisseria gonorrhoeae MsrA/B is involved in survival against reactive oxygen species.

The PilB protein of Neisseria gonorrhoeae has been reported to be involved in the regulation of pilin gene transcription, but it also possesses significant homology to the peptide methionine sulfoxide reductase family of enzymes, specifically MsrA and MsrB from Escherichia coli. MsrA and MsrB in E. coli are able to reduce methionine sulfoxide residues in proteins to methionines. In addition, the gonococcal PilB protein encodes for both MsrA and MsrB activity associated with the repair of oxidative damage to proteins. In this work, we demonstrate that the PilB protein of Neisseria gonorrhoeae is not involved in pilus expression. Additionally, we show that wild-type N. gonorrhoeae produces two forms of this polypeptide, one of which contains a signal sequence and is secreted from the bacterial cytoplasm to the outer membrane; the other lacks a signal sequence and is cytoplasmic. Furthermore, we show that the secreted form of the PilB protein is involved in survival in the presence of oxidative damage.

The mirrored methionine sulfoxide reductases of Neisseria gonorrhoeae pilB.

Methionine sulfoxide reductases (Msr) protect against oxidative damage that can contribute to cell death. The tandem Msr domains (MsrA and MsrB) of the pilB protein from Neisseria gonorrhoeae each reduce different epimeric forms of methionine sulfoxide. The overall fold of the MsrB domain revealed by the 1.85 A crystal structure shows no resemblance to the previously determined MsrA structures from other organisms. Despite the lack of homology, the active sites show approximate mirror symmetry. In each case, conserved amino acid motifs mediate the stereo-specific recognition and reduction of the substrate. Unlike the MsrA domain, the MsrB domain activates the cysteine or selenocysteine nucleophile through a unique Cys-Arg-Asp/Glu catalytic triad. The collapse of the reaction intermediate most likely results in the formation of a sulfenic or selenenic acid moiety. Regeneration of the active site occurs through a series of thiol-disulfide exchange steps involving another active site Cys residue and thioredoxin. These observations have broad implications for modular catalysis, antibiotic drug design and continuing longevity studies in mammals.

Peptide methionine sulfoxide reductase: structure, mechanism of action, and biological function.

Reactive oxygen and nitrogen intermediates can cause damage to many cellular components and have been implicated in a number of diseases. Cells have developed a variety of mechanisms to destroy these reactive molecules or repair the damage once it occurs. In proteins one of the amino acids most easily oxidized is methionine, which is converted to methionine sulfoxide. An enzyme, peptide methionine sulfoxide reductase (MsrA), catalyzes the reduction of methionine sulfoxide in proteins back to methionine. There is growing evidence that MsrA plays an important role in protecting cells against oxidative damage. This paper reviews the biochemical properties and biological role of MsrA.