Crystal structure of <i>Escherichia coli</i> DkgA, a broad‐specificity aldo‐keto reductase

Abstract

The Structural and Genomics Information Laboratory is involved in a Structural and Functional Genomics program (BIGS; http://www.igs.cnrs-mrs.fr/str_gen/) aiming at the discovery of new antibacterial targets among proteins that are ubiquitous in bacterial pathogens, exhibiting good sequence conservation, but whose precise biochemical or cellular functions remain unknown. Comprehensive bioinformatics and comparative genomics analyses were performed according to these criteria, resulting in the selection of 110 Escherichia coli candidate genes submitted to a structural genomics pipeline, the details of which have been previously described.1 Here we report the crystal structure of E. coli protein DkgA, product of the gene formerly referred to as yqhE (EcoGene accession number: EG13015). This 275-residue protein belongs to the aldo-keto reductase (AKR) family [Enzyme Commission (EC): 1.1.1.-]. AKRs constitute a group of diverse cytosolic, nicotinamide adenine dinucleotide phosphate [NAD(P)H]-dependent oxidoreductases, that catalyze the reduction of a wide range of substrates, such as aldoses, aldehydes, steroids, or monosaccharides. The precise physiological role of most of these enzymes remains unknown, although they have been collectively associated with detoxification of a broad range of substrates,2 including the toxic metabolite methylglyoxal.3 The AKR family is represented in most living cells as monomeric (α/β)8-barrel proteins about 300 amino acids in length, exhibiting a catalytic tetrad composed of a tyrosine, a lysine, an aspartate, and a histidine. Despite their common α/β-barrel fold, a conserved cofactor binding site, and the catalytic tetrad, members of the AKR family often exhibit little sequence similarity, in some cases sharing less than 10% identical residues. It has been proposed that the difference in substrate specificity exhibited by AKR family members is linked to the length and sequence of several highly variable loops in their three-dimensional (3D) structures.4 The E. coli DkgA protein was previously shown to catalyze the reduction of 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid, a key step in the biosynthesis of ascorbic acid.5 Because of its industrial interest for the production of vitamin C, the homologous enzyme (EC: 1.1.1.274) in Corynebacterium has been the target of detailed biochemical and structural studies.6-8 Subsequently, DkgA was also found to be capable of stereoselective β-keto ester reductions on ethyl acetoacetate and other 2-substituted derivatives.9 The 3D structure of the E. coli DkgA protein presented here was solved at 2.16 Å resolution by molecular replacement with the structure of the Corynebacterium homolog sharing 51% identical residues. Using NADPH as a cofactor, the recombinant DkgA protein was found to be active against a wide range of substrates, including methylglyoxal. The gene encoding DkgA was amplified from E. coli K12 genomic DNA and directional cloning was performed using the Gateway system (Invitrogen). The polymerase chain reaction (PCR) product was inserted by homologous recombination in the pDEST17 expression plasmid in phase with a N-terminal His6-tag, under the control of a T7 promoter. After transformation into DH5α cells, the purified plasmids were used for the overexpression of the recombinant proteins using our soluble expression screening protocol previously described.1 The best result was obtained with E. coli strain BL21(DE3)pLysS. Cells were grown on 2 × yeast-tryptone (2YT) medium containing ampicillin and chloramphenicol at 37°C and the pellet was resuspended in a 50 mM sodium phosphate, 300 mM NaCl buffer, pH 8.0 (buffer A), containing 0.1% Triton X-100 and 5% glycerol, before sonication. The cleared lysate was then applied to a 5 mL HiTrap Chelating Column (Pharmacia) charged with Ni2+ and equilibrated with buffer A. After sample injection, the column was washed at a flow rate of 1 mL.min−1 with 10 column volumes of buffer A, followed by 10 c