Human DJ-1 is a genetic cause of early-onset Parkinson's disease (PD), although its biochemical function is unknown. We report here that human DJ-1 and its homologs of the mouse and Caenorhabditis elegans are novel types of glyoxalase, converting glyoxal or methylglyoxal to glycolic or lactic acid, respectively, in the absence of glutathione. Purified DJ-1 proteins exhibit typical Michaelis-Menten kinetics, which were abolished completely in the mutants of essential catalytic residues, consisting of cysteine and glutamic acid. The presence of DJ-1 protected mouse embryonic fibroblast and dopaminergically derived SH-SY5Y cells from treatments of glyoxals. Likewise, C. elegans lacking cDJR-1.1, a DJ-1 homolog expressed primarily in the intestine, protected worms from glyoxal-induced death. Sub-lethal doses of glyoxals caused significant degeneration of the dopaminergic neurons in C. elegans lacking cDJR-1.2, another DJ-1 homolog expressed primarily in the head region, including neurons. Our findings that DJ-1 serves as scavengers for reactive carbonyl species may provide a new insight into the causation of PD.

We examined six Arabidopsis thaliana genes from the DJ-1/PfpI superfamily for similarity to the recently characterized bacterial and animal glyoxalases. Based on their sequence similarities, the six genes were classified into two sub-groups consisting of homologs of the human DJ-1 gene and the PH1704 gene of Pyrococcus horikoshii. Unlike the homologs from other species, all the A. thaliana genes have two tandem domains, which may have been created by gene duplication. The six AtDJ-1 proteins (a-f) were expressed in Escherichia coli for enzymatic assays with glyoxals. The DJ-1d protein, which belongs to the PH1704 sub-group, exhibits the highest activity against methylglyoxal and glyoxal, and K m values of 0.10 and 0.27 mM were measured for these two substrates, respectively, while the corresponding k cat values were 1700 and 2200 min À1 , respectively. The DJ-1a and DJ-1b glyoxalases exhibited higher specificity towards glyoxal. The other three proteins have either no or extremely low activity for glyoxals. For the DJ-1d enzyme, the residues, Cys120/313 and Glu19/212 at the active site and His121/314 and Glu94/287 at the oligomeric interface were mutated to alanines. As in other enzymes characterized to date, mutation of either the Cys or the Glu residues of the active site completely abolished enzyme activity, whereas mutation of the interface residues produced a variable decrease in activity. DJ-1d differs from its animal and bacterial homologs with respect to the configuration of its catalytic residues and the oligomeric property of the enzyme. When the wild-type DJ-1d enzyme was expressed in E. coli, the bacteria became resistant to glyoxals.

The overuse of antibiotics plays a major role in the emergence and spread of multidrug-resistant bacteria. A molecularly targeted, specific treatment method for bacterial pathogens can prevent this problem by reducing the selective pressure during microbial growth. Herein, we introduce a nonviral treatment strategy delivering genome editing material for targeting antibacterial resistance. We apply the CRISPR-Cas9 system, which has been recognized as an innovative tool for highly specific and efficient genome engineering in different organisms, as the delivery cargo. We utilize polymer-derivatized Cas9, by direct covalent modification of the protein with cationic polymer, for subsequent complexation with single-guide RNA targeting antibiotic resistance. We show that nanosized CRISPR complexes (= Cr-Nanocomplex) were successfully formed, while maintaining the functional activity of Cas9 endonuclease to induce double-strand DNA cleavage. We also demonstrate that the Cr-Nanocomplex designed to target mecA-the major gene involved in methicillin resistance-can be efficiently delivered into Methicillin-resistant Staphylococcus aureus (MRSA), and allow the editing of the bacterial genome with much higher efficiency compared to using native Cas9 complexes or conventional lipid-based formulations. The present study shows for the first time that a covalently modified CRISPR system allows nonviral, therapeutic genome editing, and can be potentially applied as a target specific antimicrobial.

DJ‐1 family proteins have recently been characterized as novel glyoxalases, although their cofactor‐free catalytic mechanisms are not fully understood. Here, we obtained crystals of Arabidopsis thaliana DJ‐1d (atDJ‐1d) and Homo sapiens DJ‐1 (hDJ‐1) covalently bound to glyoxylate, an analog of methylglyoxal, forming a hemithioacetal that presumably mimics an intermediate structure in catalysis of methylglyoxal to lactate. The deuteration level of lactate supported the proton transfer mechanism in the enzyme reaction. Differences in the enantiomeric specificity of d/l‐lactacte formation observed for the DJ‐1 superfamily proteins are explained by the presence of a His residue in the active site with essential Cys and Glu residues. The model for the stereospecificity was further evaluated by a molecular modeling simulation with methylglyoxal hemithioacetal superimposed on the glyoxylate hemithioacetal. The mechanism of DJ‐1 glyoxalase provides a basis for understanding the His residue‐based stereospecificity. Database Structural data have been submitted to the Protein Data Bank under accession numbers http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4OFW (structure of atDJ‐1d), http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4OGF (structure of hDJ‐1 with glyoxylate) and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4OGG (structure of atDJ‐1d with glyoxylate).

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