The Bacillus subtilis YkuV Is a Thiol:Disulfide Oxidoreductase Revealed by Its Redox Structures and Activity

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-XaaCys 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 thioredoxinlike fold. However, significant conformational changes in the CysXaa-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.Thioredoxins are small proteins (ϳ12 kDa) that are ubiquitously present in all kinds of life forms from archaebacteria to human (1). Generally, they function as protein thiol:disulfide oxidoreductases for maintaining the reducing environment in cytoplasm, protecting cells against hydrogen peroxide (2) and oxidative stress (3). In addition to the general function as a thiol:disulfide oxidoreductase, some thioredoxins from different organisms are also involved in various specified biological processes and therefore have specific biological functions. For example, thioredoxin is an essential subunit of bacteriophage T7 DNA polymerase in Escherichia coli (4), and in eukaryotes, thioredoxins facilitate the refolding of disulfide-containing proteins (5) and modulate the activity of certain transcription factors (6, 7). Moreover, thioredoxins have been considered as a possible target for drug development because of their roles in stimulating cancer cell growth and as an inhibitor of apoptosis (8).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). Subsequently, the structures of several thioredoxins from different species were determined (11-16). Notably, from E. coli to human, thioredoxins all share the same thioredoxin fold with the Cys-Gly-Pro-Cys active motif. In addition to thioredoxins, 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 co...

Insights into the Function of a Second, Nonclassical Ahp Peroxidase, AhpA, in Oxidative Stress Resistance in Bacillus subtilis

“…In support of this sequence analysis, studies by Zhang et al demonstrated that AhpT becomes oxidized upon interaction with oxidized AhpA in vitro, suggesting that AhpT can reduce AhpA (52). Additionally, B. subtilis thioredoxin reductase (trxB) can reduce the oxidized form of AhpT in vitro (52).…”

“…Sequence and structural analysis of AhpT clearly classifies AhpT as a CXXCcontaining, thioredoxin-like protein; compared to other thioredoxin family proteins, the sequence of AhpT clusters with the sequences of other thioredoxins from B. subtilis (Fig. 1C) (52). However, AhpT is a unique thioredoxin; it most resembles the membrane-anchored thioredoxin-like protein ResA that is important for cytochrome c biosynthesis (53).…”

“…AhpT has been characterized as a thiol-disulfide oxidoreductase on the basis of the findings of in vitro studies (52). Sequence and structural analysis of AhpT clearly classifies AhpT as a CXXCcontaining, thioredoxin-like protein; compared to other thioredoxin family proteins, the sequence of AhpT clusters with the sequences of other thioredoxins from B. subtilis (Fig.…”

Repeated triggering of sporulation in Bacillus subtilis selects against a protein that affects the timing of cell division

“…EvoI2 contains, apart from an SNP in tagE, SNPs in ykuV and yusQ. The function of yusQ is unknown, but ykuV encodes a hypothetical thiol:disulfide oxidoreductase, involved in protection of proteins against oxidative damage (Zhang et al, 2006).…”

Structural and Biophysical Analysis of the DNA Binding Properties of Myelin Transcription Factor 1

“…HADDOCK comprises a series of Python scripts that run on top of the structure determination programs ARIA (22) and CNS (23); it is designed to use biochemical and/or biophysical interaction data to generate AIRs and to then carry out docking calculations to generate models of a complex that satisfy the experimental data (37)(38)(39).…”