E-coli methionine sulfoxide reductase with a truncated N terminus or C terminus, or both, retains the ability to reduce methionine sulfoxide

The monomeric peptide methionine sulfoxide reductase (MsrA) catalyzes the i rreversible thioredoxin-dependent reduction of methionine sulfoxide. The cr ystal structure of MsrAs from Escherichia coli and Bos taurus can be descri bed as a central core of about 140 amino acids that contains the active sit e. The core is wrapped by two long N- and C-terminal extended chains. The c atalytic mechanism of the E. coli enzyme has been recently postulated to ta ke place through formation of a sulfenic acid intermediate, followed by red uction of the intermediate via intrathiol-disulfide exchanges and thioredox in 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 redu ce methionine sulfoxide in the presence of dithiothreitol. However, only th e N-terminal truncated form, which possesses the two cysteines located at t he C-terminus, reduces the sulfenic acid intermediate in a thioredoxin-depe ndent manner. The wild type displays a ping-pong mechanism with either thio redoxin or dithiothreitol as reductant. Kinetic saturation is only observed with thioredoxin with a low K-M value of 10 muM. Thus, thioredoxin is like ly the reductant in vivo. Truncations do not significantly modify the kinet ic properties, except for the double truncated form, which displays a 17-fo ld decrease in k(cat)/K-MetSO. Alternative mechanisms for sulfenic acid red uction are also presented based on analysis of available MsrA sequences.