title: Bacillus anthracis thioredoxin systems, characterization and role as electron donors for ribonucleotide reductase.
abstract: Bacillus anthracis is the causative agent of anthrax, which is associated with a high mortality rate. Like several medically important bacteria, B. anthracis lacks glutathione but encodes many genes annotated as thioredoxins, thioredoxin reductases, and glutaredoxin-like proteins. We have cloned, expressed, and characterized three potential thioredoxins, two potential thioredoxin reductases, and three glutaredoxin-like proteins. Of these, thioredoxin 1 (Trx1) and NrdH reduced insulin, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and the manganese-containing type Ib ribonucleotide reductase (RNR) from B. anthracis in the presence of NADPH and thioredoxin reductase 1 (TR1), whereas thioredoxin 2 (Trx2) could only reduce DTNB. Potential TR2 was verified as an FAD-containing protein reducible by dithiothreitol but not by NAD(P)H. The recently discovered monothiol bacillithiol did not work as a reductant for RNR, either directly or via any of the redoxins. The catalytic efficiency of Trx1 was 3 and 20 times higher than that of Trx2 and NrdH, respectively, as substrates for TR1. Additionally, the catalytic efficiency of Trx1 as an electron donor for RNR was 7-fold higher than that of NrdH. In extracts of B. anthracis, Trx1 was responsible for almost all of the disulfide reductase activity, whereas Western blots showed that the level of Trx1 was 15 and 60 times higher than that of Trx2 and NrdH, respectively. Our findings demonstrate that the most important general disulfide reductase system in B. anthracis is TR1/Trx1 and that Trx1 is the physiologically relevant electron donor for RNR. This information may provide a basis for the development of novel antimicrobial therapies targeting this severe pathogen.
authors.affiliation: Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet SE-17177 Stockholm, Sweden.
authors.forename: Tomas N
authors.initials: TN
authors.lastname: Gustafsson
authors.forename: Margareta
authors.initials: M
authors.lastname: Sahlin
authors.forename: Jun
authors.initials: J
authors.lastname: Lu
authors.forename: Britt-Marie
authors.initials: BM
authors.lastname: Sjöberg
authors.forename: Arne
authors.initials: A
authors.lastname: Holmgren
mesh_terms: D001408:Bacillus anthracis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004583:Electrons; D009249:NADP / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D012264:Ribonucleotide Reductases / Q000235:genetics / Q000378:metabolism*; D054481:Thioredoxin Reductase 1 / Q000235:genetics / Q000378:metabolism*; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D013879:Thioredoxins; D009249:NADP; D012264:Ribonucleotide Reductases; D054481:Thioredoxin Reductase 1