Novel glyoxalases from Arabidopsis thaliana

Abstract

To examine six A. thaliana (AT) genes, the DJ-1 family belonging to this family, for their similarities to the previously characterized novel glyoxalase. Based on their protein similarities, the six genes are classified into two subgroups, DJ-1 and PH1704 homologs. Unlike the members from other species, all the AT genes have repeated homology domains in a single gene, presumably forming an intramolecular dimer. For the five genes containing at least one invariant catalytic cysteine, attempts to express proteins in Escherichia coli were made after clonings of the cDNAs into appropriate vectors. The protein from DJ-1d belonging to PH1704 homologs exhibits the highest activity toward glyoxals with the Km of 0.10 mM, 0.27 mM and the kcats of 1700 min-1, 2200 min-1, respectively, while the DJ-1a and b showed considerably activity to glyoxal. The other three proteins were extremely low activity or not. For the DJ-1d, the predicted catalytic residues, the core Cys and Glu residues as well as His and Glu at the intramolecular interface, were mutated to alanines. As in other enzymes characterized so far, the changes in either Cys or Glu residue of both domains completely abolished enzymatic activity, whereas the double changes in the interface residues significantly decreased the activity. The protein is oligomeric with the size of approximately 112 kDa, corresponding to a trimer, based on data obtained with gel filtration chromatography. Configuration of the catalytic residues and oligomeric property of the DJ-1d enzyme are different from that of animals and bacteria, suggesting that the plant en-zyme is originated by creating a catalytic structure. When the wild-type DJ-1d enzyme was expressed, the strain became resistant to glyoxals, which is comparable to the resistance level exerted by the wild-type E. coli. However, effects of the catalytic mutants on enzyme activity were almost negligible.