Structural basis for the different activities of yeast Grx1 and Grx2.

Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei Anhui 230027, PR China.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 07/2010; 1804(7):1542-7. DOI: 10.1016/j.bbapap.2010.04.010
Source: PubMed

ABSTRACT Yeast glutaredoxins Grx1 and Grx2 catalyze the reduction of both inter- and intra-molecular disulfide bonds using glutathione (GSH) as the electron donor. Although sharing the same dithiolic CPYC active site and a sequence identity of 64%, they have been proved to play different roles during oxidative stress and to possess different glutathione-disulfide reductase activities. To address the structural basis of these differences, we solved the crystal structures of Grx2 in oxidized and reduced forms, at 2.10 A and 1.50 A, respectively. With the Grx1 structures we previously reported, comparative structural analyses revealed that Grx1 and Grx2 share a similar GSH binding site, except for a single residue substitution from Asp89 in Grx1 to Ser123 in Grx2. Site-directed mutagenesis in combination with activity assays further proved this single residue variation is critical for the different activities of yeast Grx1 and Grx2.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Glutathionylation plays a central role in cellular redox regulation and anti-oxidative defense. Glutaredoxins are primarily responsible reversing glutathionylation and their activity therefore affects a range of cellular processes, making them prime candidates for computational systems biology studies. However, two distinct kinetic mechanisms involving either one (monothiol) or both (dithiol) active-site cysteines have been proposed for their deglutathionylation activity and initial studies predicted that computational models based on either of these mechanisms will have different structural and kinetic properties. Further, a number of other discrepancies including the relative activity of active-site mutants and contrasting reciprocal plot kinetics have also been reported for these redoxins. Using kinetic modeling, we show that the dithiol and monothiol mechanisms are identical and, we were also able to explain much of the discrepant data found within the literature on glutaredoxin activity and kinetics. Moreover, our results have revealed how an apparently futile side-reaction in the monothiol mechanism may play a significant role in regulating glutaredoxin activity in vivo.
    Bioscience Reports 12/2014; 35(1). DOI:10.1042/BSR20140157 · 2.85 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: In biological systems, redox reactions are central to most cellular processes and the redox potential of the intracellular compartment dictates whether a particular reaction can or cannot occur. Indeed the widespread use of redox reactions in biological systems makes their detailed description outside the scope of one review. SCOPE OF THE REVIEW: Here we will focus on how system-wide redox changes can alter the reaction and transcriptional landscape of Saccharomyces cerevisiae. To understand this we explore the major determinants of cellular redox potential, how these are sensed by the cell and the dynamic responses elicited. Redox regulation is a large and complex system that has the potential to rapidly and globally alter both the reaction and transcription landscapes. Although we have a basic understanding of many of the sub-systems and a partial understanding of the transcriptional control, we are far from understanding how these systems integrate to produce coherent responses. We argue that this non-linear system self-organises, and that the output in many cases is temperature-compensated oscillations that may temporally partition incompatible reactions in vivo. Redox biochemistry impinges on most of cellular processes and has been shown to underpin ageing and many human diseases. Integrating the complexity of redox signalling and regulation is perhaps one of the most challenging areas of biology. This article is part of a Special Issue entitled Systems Biology of Microorganisms.
    Biochimica et Biophysica Acta 04/2011; 1810(10):945-58. DOI:10.1016/j.bbagen.2011.04.005 · 4.66 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Glutaredoxins (Grxs) are small oxidoreductases particularly specialized in the reduction of protein-glutathione adducts. Compared to other eukaryotic organisms, higher plants present an increased diversity of Grxs which are organized into four classes. This work presents a thorough comparative analysis of the biochemical and catalytic properties of dithiol class I Grxs from poplar, namely GrxC1, GrxC2, GrxC3, and GrxC4. By evaluating the in vitro oxidoreductase activity of wild type and cysteine mutated variants and by determining their dithiol-disulfide redox potentials, pK a values of the catalytic cysteine, redox state changes in response to oxidative treatments, two subgroups can be distinguished. In accordance with their probable quite recent duplication, GrxC1 and GrxC2 are less efficient catalysts for the reduction of dehydroascorbate and hydroxyethyldisulfide compared to GrxC3 and GrxC4, and they can form covalent dimers owing to the presence of an additional C-terminal cysteine (Cys C ). Interestingly, the second active site cysteine (CysB) influences the reactivity of the catalytic cysteine (CysA) in GrxC1 and GrxC2 as already observed with GrxC5 (restricted to A. thaliana), but not in GrxC3 and C4. However, all proteins can form an intramolecular disulfide between the two active site cysteines (CysA-CysB) which could represent either a protective mechanism considering that this second cysteine is dispensable for deglutathionylation reaction or a true catalytic intermediate occurring during the reduction of particular disulfide substrates or in specific conditions or compartments where glutathione levels are insufficient to support Grx regeneration. Overall, in addition to their different sub-cellular localization and expression pattern, the duplication and maintenance along evolution of several class I Grxs in higher plants can be explained by the existence of differential biochemical and catalytic properties.
    Frontiers in Plant Science 12/2013; 4:518. DOI:10.3389/fpls.2013.00518 · 3.64 Impact Factor