DJ-1 is a member of the large and functionally diverse DJ-1/PfpI superfamily and has homologs in nearly all organisms. Because of its connection to parkinsonism and cancer, human DJ-1 has been intensely studied for over a decade. The current view is that DJ-1 is a multifunctional oxidative stress response protein that defends cells against reactive oxygen species and mitochondrial damage, although the details of its biochemical function remain unclear. A conserved cysteine residue in DJ-1 (Cys106) is both functionally essential and subject to oxidation to the cysteine-sulfinate and cysteine-sulfonate. Consequently, the oxidative modification of Cys106 has been proposed to allow DJ-1 to act as a sensor of cellular redox homeostasis and to participate in cytoprotective signaling pathways in the cell. This review explores the current evidence for the role of cysteine oxidation in DJ-1 function, with emphasis on emerging models for how oxidative modification may regulate DJ-1's protective function and also contribute to dysfunction and disease. Antioxid. Redox Signal. 15, 111–122.

The formation of cysteine-sulfinic acid has recently become appreciated as a modification that links protein function to cellular oxidative status. Human DJ-1, a protein associated with inherited parkinsonism, readily forms cysteine-sulfinic acid at a conserved cysteine residue (Cys¹⁰⁶ in human DJ-1). Mutation of Cys¹⁰⁶ causes the protein to lose its normal protective function in cell culture and model organisms. However, it is unknown whether the loss of DJ-1 protective function in these mutants is due to the absence of Cys¹⁰⁶ oxidation or the absence of the cysteine residue itself. To address this question, we designed a series of substitutions at a proximal glutamic acid residue (Glu¹⁸) in human DJ-1 that alter the oxidative propensity of Cys¹⁰⁶ through changes in hydrogen bonding. We show that two mutations, E18N and E18Q, allow Cys¹⁰⁶ to be oxidized to Cys¹⁰⁶-sulfinic acid under mild conditions. In contrast, the E18D mutation stabilizes a cysteine-sulfenic acid that is readily reduced to the thiol in solution and in vivo. We show that E18N and E18Q can both partially substitute for wild-type DJ-1 using mitochondrial fission and cell viability assays. In contrast, the oxidatively impaired E18D mutant behaves as an inactive C106A mutant and fails to protect cells. We therefore conclude that formation of Cys¹⁰⁶-sulfinic acid is a key modification that regulates the protective function of DJ-1.

Human DJ-1, a disease-associated protein that protects cells from oxidative stress, contains an oxidation-sensitive cysteine (C106) that is essential for its cytoprotective activity. The origin of C106 reactivity is obscure, due in part to the absence of an experimentally determined pK_a value for this residue. We have used atomic-resolution X-ray crystallography and UV spectroscopy to show that C106 has a depressed pK_a of 5.4 ± 0.1 and that the C106 thiolate accepts a hydrogen bond from a protonated glutamic acid side chain (E18). X-ray crystal structures and cysteine pK_a analysis of several site-directed substitutions at residue 18 demonstrate that the protonated carboxylic acid side chain of E18 is required for the maximal stabilization of the C106 thiolate. A nearby arginine residue (R48) participates in a guanidinium stacking interaction with R28 from the other monomer in the DJ-1 dimer and elevates the pK_a of C106 by binding an anion that electrostatically suppresses thiol ionization. Our results show that the ionizable residues (E18, R48, and R28) surrounding C106 affect its pK_a in a way that is contrary to expectations based on the typical ionization behavior of glutamic acid and arginine. Lastly, a search of the Protein Data Bank (PDB) produces several candidate hydrogen-bonded aspartic/glutamic acid−cysteine interactions, which we propose are particularly common in the DJ-1 superfamily.

How changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question. Here, we use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL), ambient-temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent catalysis modulates isocyanide hydratase (ICH) conformational dynamics throughout its catalytic cycle. We visualize this previously hypothetical reaction mechanism, directly observing formation of a thioimidate covalent intermediate in ICH microcrystals during catalysis. ICH exhibits a concerted helical displacement upon active-site cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained Ile152 residue. These catalysis-activated motions permit water entry into the ICH active site for intermediate hydrolysis. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady-state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. These results demonstrate that MISC can capture otherwise elusive aspects of enzyme mechanism and dynamics in microcrystalline samples, resolving long-standing questions about the connection between nonequilibrium protein motions and enzyme catalysis.

Methylglyoxal is a cytotoxic reactive carbonyl compound produced by central metabolism. Dedicated glyoxalases convert methylglyoxal to d-lactate using multiple catalytic strategies. In this study, the DJ-1 superfamily member ORF 19.251/GLX3 from Candida albicans is shown to possess glyoxalase activity, making this the first demonstrated glutathione-independent glyoxalase in fungi. The crystal structure of Glx3p indicates that the protein is a monomer containing the catalytic triad Cys(136)-His(137)-Glu(168). Purified Glx3p has an in vitro methylglyoxalase activity (Km = 5.5 mM and kcat = 7.8 s(-1)) that is significantly greater than that of more distantly related members of the DJ-1 superfamily. A close Glx3p homolog from Saccharomyces cerevisiae (YDR533C/Hsp31) also has glyoxalase activity, suggesting that fungal members of the Hsp31 clade of the DJ-1 superfamily are all probable glutathione-independent glyoxalases. A homozygous glx3 null mutant in C. albicans strain SC5314 displays greater sensitivity to millimolar levels of exogenous methylglyoxal, elevated levels of intracellular methylglyoxal, and carbon source-dependent growth defects, especially when grown on glycerol. These phenotypic defects are complemented by restoration of the wild-type GLX3 locus. The growth defect of Glx3-deficient cells in glycerol is also partially complemented by added inorganic phosphate, which is not observed for wild-type or glucose-grown cells. Therefore, C. albicans Glx3 and its fungal homologs are physiologically relevant glutathione-independent glyoxalases that are not redundant with the previously characterized glutathione-dependent GLO1/GLO2 system. In addition to its role in detoxifying glyoxals, Glx3 and its close homologs may have other important roles in stress response.