Thioredoxin-h1 Reduces and Reactivates the Oxidized Cytosolic Malate Dehydrogenase Dimer in Higher Plants

Abstract

Cytosolic malate dehydrogenase (cytMDH) was captured by thioredoxin affinity chromatography as a possible target protein of cytosolic thioredoxin (Yamazaki, D., Motohashi, K., Kasama, T., Hara, Y., and Hisabori, T. (2004) Plant Cell Physiol. 45, 18 -27). To further dissect this interaction, we aimed to determine whether cytMDH can interact with the cytosolic thioredoxin and whether its activity is redox-regulated. We obtained the active recombinant cytMDH that could be oxidized and rendered inactive. Inactivation was reversed by incubation with low concentrations of dithiothreitol in the presence of recombinant Arabidopsis thaliana thioredoxin-h1. Inactivation of cytMDH was found to result from formation of a homodimer. By cysteine mutant analysis and peptide mapping analysis, we were able to determine that the cytMDH homodimer occurs by formation of a disulfide bond via the Cys 330 residue. Moreover, we found this bond to be efficiently reduced by the reduced form of thioredoxin-h1. These results demonstrate that the oxidized form cytMDH dimer is a preferable target protein of the reduced form thioredoxin-h1 as suggested by thioredoxin affinity chromatography. Malate dehydrogenase (MDH)3 catalyzes the reversible reaction of oxaloacetate to malate utilizing the NAD ϩ /NADH or NADP ϩ /NADPH cofactor system and is known to occur ubiquitously in mammals, plants, and most prokaryotes. NAD-dependent MDHs (EC 1.1.1.37) generally exist as a homodimer with a subunit of 32-37 kDa (1, 2), whereas NADP-dependent MDH (EC 1.1.1.82) possesses a larger subunit of ϳ42 kDa (3, 4). In plants, MDH isoforms have been reported in the cytosol and in organelles and have been found to carry out important roles in a number of metabolic pathways (5). In general, MDH proteins are grouped into five classes according to their location within the cell; (i) cytosolic NAD-dependent MDH (cytMDH); (ii) mitochondrial NAD-dependent MDH, which is part of the tricarboxylic acid cycle; (iii) glyoxisomal and peroxisomal NAD-dependent MDHs, which are involved in photorespiration; (iv) chloroplast NADPdependent MDH, which is required for the transfer of reducing equivalents from chloroplast stroma to cytosol; and (v) chloroplast NAD-dependent MDH (6).Among the classes listed, chloroplast NADP-dependent MDH is a thiol enzyme that has been subject of intense study (4,(7)(8)(9)(10)(11)(12) and that uses NADP ϩ for catalysis. Chloroplast NADPdependent MDH possesses both N-and C-terminal extensions, both of which harbor a redox-sensitive cysteine pair (4,(12)(13)(14). Central to the regulation of chloroplast MDH, ferredoxin-thioredoxin reductase sources electrons from photosynthesis to reduce chloroplast thioredoxins (Trx) (8). Trxs are an important class of regulatory proteins that modulate the activity of a wide variety of thiol-containing chloroplast enzymes by the reduction of specific disulfide bonds (15). In the light, excess NADPH (produced by photosynthesis but surplus to CO 2 fixation requirements) induces the conversion of oxaloacetate to malate, r...

From climate change to molecular response: redox proteomics of ozone‐induced responses in soybean

“…Moreover, phosphoribulokinase and GAPDH interact via thioredoxin-mediated redox changes in response to light intensity (Howard et al, 2008), which may be affected by O 3 -related changes in oxidation state. Similarly, MDH is a critical regulatory point in the citric acid cycle; however, the cytosolic form of the enzyme is redox-regulated and inactivated under oxidizing conditions (Hara et al, 2006). In the high-O 3 leaf sample, MDH had two and fivefold higher expression and oxidation compared with controls with a modest 1.3-fold increase in total activity (Table 2).…”

Heat-Shock and Redox-Dependent Functional Switching of an h-Type Arabidopsis Thioredoxin from a Disulfide Reductase to a Molecular Chaperone

“…However, in contrast to E. coli Trx, which is reported to refold partially denatured substrate proteins (Kern et al, 2003), AtTrx-h3 did not exhibit refolding activity of urea-denatured MDH as a substrate (data not shown). On the other hand, it was recently reported that the Trx-h1 efficiently reduced and reactivated the oxidized form of cytosolic MDH in higher plants (Hara et al, 2006). Because the inactivation of important intracellular enzyme by oxidation, such as cytosolic MDH, results in a marked impairment of cell viability, Trx-h1 can act as a redox-sensitive reducer of the protein accompanying its structural transition.…”

Quantifying changes in the thiol redox proteome upon oxidative stress in vivo

“…Deletion of either the mdh gene or the yggX gene was found to cause an increase in both H 2 O 2 and NaOCl stress sensitivity, indicating that both proteins generally protect E. coli against oxidative stress. Interestingly, a NAD ϩ -dependent MDH homologue from higher plants has recently been identified as a thioredoxin substrate (33). It is therefore tempting to speculate that the abundant NAD ϩ -dependent E. coli MDH might exert oxidative stress protection by increasing the in vivo turnover of oxidants using thioredoxindependent redox-cycling.…”