May 2012 featured article
A Pseudomonas L-serine dehydrogenase
Structural studies of an uncharacterized Pseudomonas aeruginosa enzyme reveal a dehydrogenase with a substrate preference for L-serine.
Structure of the PA0743 tetramer, with each chain colored differently. The modeled HEPES is shown in stick representation. Figure courtesy of Alexander Yakunin.
The β-hydroxyacid dehydrogenases constitute a ubiquitous family of enzymes. Some members are involved in central metabolic processes in various organisms, but the vast majority remains uncharacterized.
In a recent report, Yakunin and colleagues (PSI MCSG) performed structural studies on the predicted β-hydroxyisobutyrate dehydrogenase PA0743 from the bacterium Pseudomonas aeruginosa, which encodes six predicted members of the enzyme family. The preferred substrates of affinity-purified PA0743 are L-serine and methyl-DL-serine (not the predicted substrate, 3-hydroxyisobutyrate). The enzyme catalyzes cofactor-dependent oxidation of the substrate hydroxyl acid to a semialdehyde; its preferred cofactor is NAD+, but NADP+ can also be used, although yielding lower activity.
The authors solved the crystal structure of the apoenzyme (PDB 3OBB) to 2.2 Å, revealing a two-domain protein with an N-terminal Rossmann fold domain and a C-terminal all-α domain connected by a long α9 helix. Consistent with gel filtration studies, structural analysis indicated that the enzyme is tetrameric. The researchers observed a weak electron density in the interdomain cleft close to the predicted active site that was modeled as a HEPES molecule, as HEPES was present in the crystallization buffer. They predicted that HEPES binding mimics that of the L-serine or methyl-DL-serine substrate.
The crystal structure of the PA0743-NAD+ complex (PDB 3Q3C) revealed that NAD+ also binds within the interdomain cleft at a non-overlapping site with HEPES. NAD+ binding does not cause major structural rearrangements relative to the apoenzyme. Site-directed mutagenesis confirmed the critical role of the active site lysine, Lys171, and defined additional residues important for NAD+-dependent L-serine dehydrogenase activity.
The biological function of PA0743 in P. aeruginosa remains unclear. While its substrate preference suggests an involvement in serine metabolism, Yakunin and colleagues observed that mutant strains can grow on L-serine as a sole nitrogen source. It is possible that other enzymes may compensate, particularly under laboratory conditions, or PA0743 may be involved in other metabolic processes. Further experiments will be needed to elucidate its biological role, and will doubtless be aided by continued structural studies on proteins of this interesting enzyme family.