Structure-guided approach to modify the substrate specificity of the protein human deglycase-1 (hDJ-1).

Glycation is a non-enzymatic reaction wherein sugars or dicarbonyls such as methylglyoxal (MGO) and glyoxal (GO) react with proteins, leading to protein inactivation. The hydrolysing enzyme human deglycase-1 (hDJ-1) is reported to decrease glycative stress by deglycating the modified proteins, specifically at cysteine, lysine, and arginine sites. This specificity of hDJ-1 is thought to be regulated by its active site cysteine residue (Cys106). Structural analysis of hDJ-1 by molecular docking and simulation studies, however, indicates a possible role of glutamate (Glu18) in determining its substrate specificity. To elucidate this, Glu18 present at the catalytic site of hDJ-1 was modified to aspartate (Asp18) by SDM, and the resultant mutant was termed mutant DJ-1 (mDJ-1). Both hDJ-1 and mDJ-1 were heterologously expressed in Escherichia coli BL21 (DE3) strain and purified to homogeneity. The hDJ-1 showed k values of 1.45 × 10 s, 3.6 × 10 s, and 3.1 × 10 s, and K values 0.181 mM, 18.18 mM, and 12.5 mM for N-acetylcysteine (NacCys), N-acetyllysine (NacLys), and N-acetylarginine (NacArg), respectively. The mDJ-1 showed altered k values (8 × 10 s, 3.8 × 10 s, 4.9 × 10 s) and K values of 0.14 mM, 6.25 mM, 5.88 mM for NacCys, NacLys and NacArg, respectively. A single amino acid change (Glu18 to Asp18) improved the substrate specificity of mDJ-1 toward NacLys and NacArg. Understanding hDJ-1's structure and enhanced functionality will facilitate further exploration of its therapeutic potential for the treatment of glycation-induced diabetic complications.