Abstract

The Bacillus subtilis YkuV responds to environmental oxidative stress and plays an important role for the bacteria to adapt to the environment. Bioinformatic analysis suggests that YkuV is a homolog of membrane-anchored proteins and belongs to the thioredoxin-like protein superfamily containing the typical Cys-Xaa-Xaa-Cys active motif. However, the biological function of this protein remains unknown thus far. In order to elucidate the biological function, we have determined the solution structures of both the oxidized and reduced forms of B. subtilis YkuV by NMR spectroscopy and performed biochemical studies. Our results demonstrated that the reduced YkuV has a low midpoint redox potential, allowing it to reduce a variety of protein substrates. The overall structures of both oxidized and reduced forms are similar, with a typical thioredoxin-like fold. However, significant conformational changes in the Cys-Xaa-Xaa-Cys active motif of the tertiary structures are observed between the two forms. In addition, the backbone dynamics provide further insights in understanding the strong redox potential of the reduced YkuV. Furthermore, we demonstrated that YkuV is able to reduce different protein substrates in vitro. Together, our results clearly established that YkuV may function as a general thiol:disulfide oxidoreductase, which acts as an alternative for thioredoxin or thioredoxin reductase to maintain the reducing environment in the cell cytoplasm.

Highlights

  • The thioredoxin fold was first characterized from E. coli thioredoxin, which consists of a central five-stranded mixed ␤-sheet surrounded by four ␣-helices (9 –11)

  • A large number of proteins comprising the basic thioredoxin fold with the CysXaa-Xaa-Cys active motif in the active site have been characterized as thioredoxin-like proteins, which consist of a central four-stranded mixed ␤-sheet surrounded by three ␣-helices [17, 18]

  • Sequence analysis revealed that there are at least seven genes in B. subtilis genome encoding cytoplasmic thioredoxin-like proteins [3], which leads to an open question: are any products of these genes engaged in these biological processes?

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Summary

The abbreviations used are

TrxA, thioredoxin; TrxB, thioredoxin reductase; ArsC, arsenate reductase; HSQC, heteronuclear single quantum coherence; DTT, 1,4-dithiothreitol; DTNB, 5,5Ј-dithiobis-2-nitrobenzoic acid; r.m.s.d., root mean square deviation; CMP, cytochrome maturation protein; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; HSQC, heteronuclear single quantum coherence; TOCSY, total correlation spectroscopy. We have characterized the backbone dynamic properties of this protein, which provide further insights in understanding the low redox potential of the reduced YkuV. These results in conjunction with the in vitro experiments strongly suggest that YkuV may function as a general thiol:disulfide oxidoreductase in vivo

EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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