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UMR CNRS-UHP 7567, Maturation des ARN et Enzymologie Moléculaire, Faculté des Sciences, Bld des Aiguillettes, 54506 Vandoeuvre-les-Nancy, France
Reprint requests to: Guy Branlant, UMR CNRS-UHP 7567, Maturation des ARN et Enzymologie Moléculaire, Faculté des Sciences, Bld des Aiguillettes, BP 239, 54506 Vandoeuvre-les-Nancy, France; e-mail: Guy.Branlant{at}maem.uhp-nancy.fr; fax: (33) 3-83-91-20-93.
The monomeric peptide methionine sulfoxide reductase (MsrA) catalyzes the irreversible thioredoxin-dependent reduction of methionine sulfoxide. The crystal structure of MsrAs from Escherichia coli and Bos taurus can be described as a central core of about 140 amino acids that contains the active site. The core is wrapped by two long N- and C-terminal extended chains. The catalytic mechanism of the E. coli enzyme has been recently postulated to take place through formation of a sulfenic acid intermediate, followed by reduction of the intermediate via intrathiol-disulfide exchanges and thioredoxin oxidation. In the present work, truncated MsrAs at the N- or C-terminal end or at both were produced as folded entities. All forms are able to reduce methionine sulfoxide in the presence of dithiothreitol. However, only the N-terminal truncated form, which possesses the two cysteines located at the C-terminus, reduces the sulfenic acid intermediate in a thioredoxin-dependent manner. The wild type displays a ping-pong mechanism with either thioredoxin or dithiothreitol as reductant. Kinetic saturation is only observed with thioredoxin with a low KM value of 10 µM. Thus, thioredoxin is likely the reductant in vivo. Truncations do not significantly modify the kinetic properties, except for the double truncated form, which displays a 17-fold decrease in kcat/KMetSO. Alternative mechanisms for sulfenic acid reduction are also presented based on analysis of available MsrA sequences.
Abbreviations: DTNB, 5,5'-dithiobis(2-nitrobenzoate) • DTT, dithiothreitol • KDTT, KM for DTT • KMetSO, KM for MetSO • MetSO, methionine sulfoxide • MsrA, methionine sulfoxide reductase • TFA, trifluoroacetic acid • TNB-, 3-carboxy-4-nitrobenzenethiol • Trx, thioredoxin
Keywords: Methionine sulfoxide reductase; truncated enzymes; sulfenic acid reduction; thioredoxin
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