Reaction mechanism, evolutionary analysis, and role of zinc in Drosophila methionine-R-sulfoxide reductase

R. Abhilash Kumar, Ahmet Koc, Ronald Cerny, Vadim N. Gladyshev

Research output: Contribution to journalArticle

129 Citations (Scopus)

Abstract

Methionine residues in proteins are susceptible to oxidation, and the resulting methionine sulfoxides can be reduced back to methionines by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). Herein, we have identified two MsrB families that differ by the presence of zinc. Evolutionary analyses suggested that the zinc-containing MsrB proteins are prototype enzymes and that the metal was lost in certain MsrB proteins later in evolution. Zinc-containing Drosophila MsrB was further characterized. The enzyme was found to employ a catalytic Cys124 thiolate, which directly interacted with methionine sulfoxide, resulting in methionine and a Cys124 sulfenic acid intermediate. A subsequent reaction of this intermediate with Cys69 generated an intramolecular disulfide. Dithiothreitol could reduce either the sulfenic acid or the disulfide, but the disulfide was a preferred substrate for thioredoxin, a natural electron donor. Interestingly, the C69S mutant could complement MsrA/MsrB deficiency in yeast, and the corresponding natural form of mouse MsrB was active with thioredoxin. These data indicate that MsrB proteins employ alternative mechanisms for sulfenic acid reduction. Four other conserved cysteines in Drosophila MsrB (Cys51, Cys54, Cys101, and Cys104) were found to coordinate structural zinc. Mutation of any one or a combination of these residues resulted in complete loss of metal and catalytic activity, demonstrating an essential role of zinc in Drosophila MsrB. In contrast, two conserved histidines were important for thioredoxin-dependent activity, but were not involved in zinc binding. A Drosophila MsrA gene was also cloned, and the recombinant enzyme was found to be metal-free and specific for methionine S-sulfoxide and to employ a similar sulfenic acid/disulfide mechanism.

Original languageEnglish (US)
Pages (from-to)37527-37535
Number of pages9
JournalJournal of Biological Chemistry
Volume277
Issue number40
DOIs
StatePublished - Oct 4 2002

Fingerprint

Methionine Sulfoxide Reductases
Sulfenic Acids
Methionine
Drosophila
Zinc
Disulfides
Thioredoxins
Metals
sulfoxide
Proteins
Enzymes
Dithiothreitol
Histidine
Cysteine
Yeast
Yeasts
Catalyst activity
Electrons
Genes
Mutation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Reaction mechanism, evolutionary analysis, and role of zinc in Drosophila methionine-R-sulfoxide reductase. / Abhilash Kumar, R.; Koc, Ahmet; Cerny, Ronald; Gladyshev, Vadim N.

In: Journal of Biological Chemistry, Vol. 277, No. 40, 04.10.2002, p. 37527-37535.

Research output: Contribution to journalArticle

Abhilash Kumar, R. ; Koc, Ahmet ; Cerny, Ronald ; Gladyshev, Vadim N. / Reaction mechanism, evolutionary analysis, and role of zinc in Drosophila methionine-R-sulfoxide reductase. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 40. pp. 37527-37535.
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