Biochemical and Structural Studies of Uncharacterized Protein PA0743 from Pseudomonas aeruginosa Revealed

The-hydroxyacid dehydrogenases form a large family of ubiquitous enzymes that catalyze oxidation of various -hydroxy acid substrates to corresponding semialdehydes. Several known enzymes include -hydroxyisobutyrate dehydrogenase, 6-phosphogluconate dehydrogenase, 2-(hydroxymethyl)glutarate dehydrogenase, and phenylserine dehydrogenase, but the vast majority of -hydroxyacid dehydrogenases remain uncharacterized. Here, we demonstrate that the predicted-hydroxyisobutyrate dehydrogenase PA0743 from Pseudomonas aeruginosa catalyzes an NAD-dependent oxidation of L-serine and methyl-L-serine but exhibits low activity against -hydroxyisobutyrate. Two crystal structures of PA0743 were solved at 2.2–2.3-A resolution and revealed an N-terminal Rossmann fold domain connected by a long -helix to the C-terminal all- domain. The PA0743 apostructure showed the presence of additional density modeled as HEPES bound in the interdomain cleft close to the predicted catalytic Lys171, 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