Diechtierow, M.

Feher, K.

Füller, F.

Krauth-Siegel, R.L.

Melchers, J.

Muhle-Goll, C.

Tews, I.

Ulrich, A.S.

English

Ulrich, A. S.

Krauth-Siegel, R. L.

KITopen

The strictly conserved Cys76 plays a crucial role in the conformational stability of reduced Px IIIConclusions: Cys76 was regarded as unimportant for the catalytic reaction (Schlecker et al., 2007). It is, however, conserved throughout the entire family of selenocysteine and cysteine glutathione peroxidases. Our results show that itplays a critical role for the reduced enzyme in assuming different conformational states. This implies that the catalytic triad may only be required as short-lived intermediate during catalysis and that Cys76 influences the equilibrium between different conformations. References: Schlecker T, Comini MA, Melchers J, Ruppert T, Krauth-Siegel RL. (2007) Biochem J. 405:445-54.; Melchers J, Diechtierow M, Fehér K, Sinning I, Tews I, Krauth-Siegel RL, Muhle-Goll C. (2008) J Biol Chem 283:30401-11. Alphey MS, König J, Fairlamb AH. (2008) Biochem J. 414:375-81.; Muhle-Goll C, Füller F, Ulrich AS, Krauth-Siegel RL. (2010) FEBS Lett. 584:1027-32. Introduction: In trypanosomes and leishmania are the causative agents of several tropical diseases. The parasites lack catalases and selenocysteine-containing gluthathione peroxidases. Instead, cysteine-containing glutathione peroxidase-type (Px I, Px II, and Px III) enzymes are – together with 2-Cys-peroxiredoxins - responsible for hydroperoxide reduction acting both as tryparedoxin peroxidases. Px III belongs to a subgroup of glutathione peroxidase-type enzymes, in which the catalytic selenocysteine is replaced by a cysteine. In classical selenocysteine glutathione peroxidases, the reduction of the peroxide is achieved through attack by the selenocysteine, which subsequently is reduced by glutathione. In the cysteine homologues, a second resolving cysteine takes over the part of the glutathione and forms an intermediate disulfide bridge. In T. brucei, the glutathione peroxidase-type enzymes proved to be essential. Unravelling their mechanism could provide a basis to control the parasite.Reaction scheme of Px IIIWith NADPH as primary electron source, the reducing equivalents flow via trypanothione and tryparedoxin (Tpx), a distant relative of the thioredoxin protein family, onto the peroxidases, which then reduce the hydroperoxide substrates. Structure determination by NMR and X-ray crystallographyClaudia Muhle-Golla,b,c , Johannes Melchersa,d, Michael Diechtierowd, Florian Füllerd, Anne. S. Ulrichc, Krisztina Fehera,b, Ivo Tewsd, R. Luise Krauth-SiegeldaEMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany; bMax-Planck-Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany; cKarlsruheInstitute of Technology, Campus Nord, Institut für biologische Grenzflächen (IBG-2), POB 3640, 76021 Karlsruhe, Germany, d Biochemie-Zentrum, UniversitätHeidelberg, Im Neuenheimer Feld 504, 69120 Heidelberg, Germanyoxidized C76S Px III,  X-ray structure oxidized C76S Px III NMR-structurereduced C76S Px III, NMR structureThe two catalytically active cysteine residues Cys47 on loop L1 and Cys95 on loop L2 are depicted in orange. The reduced and the oxidized form of C76S mutant of  Px III are almost identical. Contrary to our results, the crystal structure of Px IIpublished by the group of Fairlamb (right) shows the presence of a catalytic triad in the reduced form (W129, Q74, C39) and an additional helix like the crystal structures of classical glutathione peroxidases (Alphey2008). Note the large distance between the Cysteines in Px II (green arrows). Difference attributable to method or mutation C76S?Comparison of the reduced structures of C76S Px III with Px II NMR structure of C76S Px III (Melchers et al., 2008)90°0°The loop L2 harbouring Cys95 shows conformational exchange and has little NOE contacts under both conditions.A BHetNOE reporting on dynamics of the proteinNMR structure ensemble (A) oxidized and (B) reduced protein. Residues showing conformational exchange are depicted in green.oxidizedreducedComparison of WT and C76S mutantCD spectra reveal different increase in helicity upon reduction.Trp fluorescence spectra show that the environment of Trp137 changes differently upon reduction.15N-HSQC spectra corroborate that Trp137 is different in WT and C76S mutant Selectively labelled with 15N-TrpUniformly 15N-labelledCys76 appears to influence the equilibrium between different conformations. *oxidizedReduction with DTT5 min16 min54 min30 min*oxidizedReduction with DTT5 min14 min32 min23 min10 mM H2O2*oxidizedReduction with DTT4 min15 min36 min24 minxoxidizedReduction with DTT4 min36 min24 minx10 mM H2O215 min*oxidized reducedWT C76SWT C76STrypanosomes and leishmania have a unique thiol redox metabolism that is based on trypanothione [N1,N8-bis(glutathionyl)spermidine] and the flavoenzyme trypanothione reductase.Crystal structure of Px II (Alphey et al., 2008)oxidizedPx IIIreduced Px IIIblack WTred C76Sblack WTred C76Sblack WToxred WTredblue C76Soxyellow C76Sred

The strictly conserved Cys76 plays a crucial role in the conformational stability of reduced Px III

https://publikationen.bibliothek.kit.edu/220080558/3815747

The strictly conserved Cys76 plays a crucial role in the conformational stability of reduced Px III

Abstract

Similar works

Full text

Available Versions

KITopen