Abstract
The β-hydroxyacid dehydrogenases form a large family of ubiquitous enzymes that catalyze oxidation of various β-hydroxy acid substrates to corresponding semialdehydes. Several known enzymes include β-hydroxyisobutyrate dehydrogenase, 6-phosphogluconate dehydrogenase, 2-(hydroxymethyl)glutarate dehydrogenase, and phenylserine dehydrogenase, but the vast majority of β-hydroxyacid dehydrogenases remain uncharacterized. Here, we demonstrate that the predicted β-hydroxyisobutyrate dehydrogenase PA0743 from Pseudomonas aeruginosa catalyzes an NAD(+)-dependent oxidation of l-serine and methyl-l-serine but exhibits low activity against β-hydroxyisobutyrate. Two crystal structures of PA0743 were solved at 2.2-2.3-Å resolution and revealed an N-terminal Rossmann fold domain connected by a long α-helix to the C-terminal all-α domain. The PA0743 apostructure showed the presence of additional density modeled as HEPES bound in the interdomain cleft close to the predicted catalytic Lys-171, revealing the molecular details of the PA0743 substrate-binding site. The structure of the PA0743-NAD(+) complex demonstrated that the opposite side of the enzyme active site accommodates the cofactor, which is also bound near Lys-171. Site-directed mutagenesis of PA0743 emphasized the critical role of four amino acid residues in catalysis including the primary catalytic residue Lys-171. Our results provide further insight into the molecular mechanisms of substrate selectivity and activity of β-hydroxyacid dehydrogenases.
Highlights
The -hydroxyacid dehydrogenases form a large family of ubiquitous enzymes that catalyze oxidation of various -hydroxy acid substrates to corresponding semialdehydes
PA0743 shares 32.9 –38.2% sequence identity with the biochemically characterized HIBA dehydrogenases from rat and T. thermophilus (Fig. 1)
PA0743 shows rather high similarity (62% sequence identity) to PA3569, which has been proposed to be a HIBA dehydrogenase in P. aeruginosa (Fig. 1) [21]. This level of sequence similarity between PA0743 and PA3569 suggests that PA0743 might be another HIBA dehydrogenase in P. aeruginosa
Summary
The assays were performed at 37 °C in a reaction mixture (1 ml) containing 50 mM diethanolamine buffer (pH 11.0), 5 mM NADϩ (or NADPϩ), 5–15 mM substrate, and 1–10 g of PA0743. Plasmids containing the desired mutations were transformed into the E. coli BL21(DE3) strain, and the mutant PA0743 proteins were overexpressed and purified in the same manner as the wild-type PA0743. The strains were grown aerobically at 37 °C (250 rpm) on MOPS minimal medium supplemented with glucose (0.2%) [27] and containing L-serine or other substrates as nitrogen sources (leucine, valine, uracil, and thymine; 1 g/liter). The final models were refined to an Rwork and Rfree of 19.7 and 24.8% for the apostructure and 19.1 and 26.2% for the structure of PA0743-NAD complex, respectively. Protein Data Bank Accession Codes—Coordinates and structure factors have been deposited under accession codes 3OBB (apostructure) and 3Q3C (NADϩ complex)
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