Characterization and Manipulation of the Pseudomonas aeruginosa Dimethylarginine Dimethylaminohydrolase Monomer–Dimer Equilibrium

In mammals, the enzyme dimethylarginine dimethylaminohydrolase (DDAH) is implicated in the regulation of the cellular levels of asymmetric methylarginines, small molecule metabolites that themselves represent a family of endogenous inhibitors of nitric oxide synthase (NOS). The involvement of DDAH function in the regulation of NOS makes this enzyme a potentially attractive therapeutic target. DDAH from the bacterium Pseudomonas aeruginosa (PaDDAH) is so far the only structurally tractable homologue of mammalian DDAH isoforms. To complement the recent crystal structure of this protein, we show by hydrodynamic measurements that PaDDAH exists in dynamic equilibrium between monomer (ca 29 kDa) and symmetric homodimer (ca 58 kDa) states with a dimer dissociation constant, Kd∼500 nM. For the purposes of NMR-based approaches to the study of this enzymeʹs interactions with substrate and inhibitor ligands, it would be useful to obtain the protein in monomeric form. Through detailed analysis of the homodimer PaDDAH crystal structure we identified key residues involved in the protomer–protomer interface and targeted these for mutation. The hydrodynamic and self-associative properties of a series of PaDDAH interface mutants were analyzed by concentration-dependent analytical size-exclusion chromatography and sedimentation equilibrium analytical ultracentrifugation. The individual substitution of several of the interface residues shifts the equilibrium position towards the monomer, which allowed the design of a double mutant variant (Arg40→Glu, Arg98→His) that behaves exclusively as a stable monomer, yet retains greater than 95% catalytic activity compared to wild-type. Comparative two-dimensional 1H, 15N heteronuclear NMR spectra indicate that the double mutant remains a monomer even at ∼1 mM concentration. Accordingly, the double mutant PaDDAH is an attractive template for further NMR-based investigations of the enzyme mechanism and characterization of ligand-binding and inhibitor-binding profiles. These results indicate that dimerization of PaDDAH is not critical for the maintenance of the biological function of the protein. These results are discussed in the context of known modes of self-association between structurally related, but functionally distinct, members of the β/α-propeller fold class.