Biochemical and structural studies of uncharacterized protein PA0743 from Pseudomonas aeruginosa revealed NAD+-dependent L-serine dehydrogenase

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.

FIGURE 1.

Structure-based sequence alignment of PA0743 and several β-hydroxyacid dehydrogenases. The secondary structure elements of PA0743 and human HIBA dehydrogenase are shown above and below the alignment, respectively. Residues conserved in all aligned β-hydroxyacid dehydrogenases are boxed. The residues comprising the four characteristic β-hydroxyacid dehydrogenase sequence motifs (3) are highlighted in different colors and labeled below the alignment. The PA0743 residues mutated to Ala in this work are marked with an asterisk above the alignment and numbered. The proteins compared are PA0743 (UniProTKB Q9I5I6), PA3569 (P28811), rat HIBA dehydrogenase (P29266), the HIBA dehydrogenase TTHA0237 from T. thermophilus (Q5SLQ6), 2-(hydroxymethyl)glutarate dehydrogenase Hgd from E. barkeri (Ebar) (Q0QLF5), and the predicted human HIBA dehydrogenase (HIBADH) (P31937).

FIGURE 2.

Substrate profile of PA0743. Dehydrogenase activity of purified PA0743 against l-serine and other substrates measured in the presence of 5 mm NAD⁺ (as described under “Experimental Procedures”) is shown. The following substrates were also used for screening and produced negative results: (S)-l-β-hydroxybutyric acid, (R)-(−)-3-hydroxybutyric acid, 2-hydroxyisobutyric acid, methyl-2-hydroxyisobutyric acid, ethyl-2-hydroxyisobutyric acid, 2-hydroxybutyric acid, dl-malic acid, and dl-homoserine. M(R)-3H-2M-Propionate, methyl-(R)-3-hydroxy-2-methylpropionic acid; M-2,2dM-3H-Propionate, methyl-2,2-dimethyl-3-hydroxypropionic acid; tertB-3H-Propionate, tert-butyl-3-hydroxypropionic acid; M(S)-3H-2M-Propionate, methyl-(S)-3-hydroxy-2-methylpropionic acid. Each bar represents an average of the results from at least two independent determinations, with S.D. indicated by error bars (in all figures).

FIGURE 3.

Proposed reactions catalyzed by PA0743. The enzyme catalyzes the NAD⁺-dependent dehydrogenation of l-serine (A) or methyl-l-serine (B) at C3, producing 2-aminomalonate semialdehyde or 2-aminomethylmalonate semialdehyde, respectively, and NADH.

FIGURE 4.

Crystal structure of PA0743: overall structure of protomer, dimer, and tetramer. A, the protomer structure is shown in three orientations (related by 90° rotation) with the N-terminal domain colored orange, the C-terminal domain in cyan, and the long α9 helix connecting the two domains in green. The N and C termini are labeled (N and C). B, the dimer structure is shown in two orientations (related by 90° rotation) with the protomers colored in orange and green. C, the PA0743 tetramer with four protomers shown in different colors (cyan, magenta, orange, and green).

FIGURE 5.

Close-up view of PA0743 structure with bound HEPES or NAD⁺. A and B, surface presentation of the PA0743 active site with the modeled HEPES or bound NAD⁺. The substrate-binding site (A) is formed by the residues from two protomers (from one dimer) shown in different colors (gray and tan), whereas the cofactor-binding site (B) is made of the residues from one protomer. Both ligands are almost completely buried in the PA0743 active site. The location of the cofactor- and substrate-binding sites is labeled (NAD site and Substrate site with an arrow, respectively). C and D, electron density maps showing the positions of bound HEPES (C) or NAD⁺ (D) and selected residues (labeled). The omit map was generated by omitting the HEPES (C) and NAD⁺ (D) from the model. Cyan-colored density represents the resulting F_o − F_c map contoured at 3.0 σ. Ligand atoms and selected PA0743 residues are labeled and shown as a stick representation with magenta carbon atoms representing the ligand and green carbon atoms representing the selected PA0743 residues.

FIGURE 6.

Close-up stereoview of PA0743 active site. A, substrate-binding site with the modeled HEPES molecule. B, cofactor-binding site with the bound NAD⁺. C, the relative orientation of NAD⁺ and HEPES near the catalytic Lys-171 in the active site. Two PA0743 structures (Protein Data Bank codes 3OBB and 3Q3C) were superimposed, and the PA0743 ribbon from 3OBB is shown with the bound NAD⁺ and HEPES. The amino acid side chains and ligands are shown as sticks and labeled along with the protein ribbon of two protomers colored in gray and tan.

FIGURE 7.

Alanine replacement mutagenesis of PA0743. Dehydrogenase activity of purified mutant proteins with l-serine as substrate (in the presence of 5 mm NAD⁺) is shown.