An NMR investigation of the structure-function characteristics of the Pseudomonas aeruginosa dimethylarginine dimethylaminohydrolase
The enzyme dimethylarginine dimethylaminohydrolase (DDAH) is responsible for the metabolism of free asymmetric methylarginine residues (AMAs) to citrulline and mono- or di-methylamines. In mammals, the asymmetric methylarginines NN11-dimethylarginine (ADMA) and Nn-methylarginine (L-NMMA) are endogenous inhibitors of all nitric oxide synthase (NOS) isoforms. By controlling local levels of AMAs, DDAH activity is implicated in the regulation of nitric oxide (NO) production, which gives rise to interest in the therapeutic potential of this enzyme. This work describes the application of NMR methodology to study the backbone dynamics of PaDDAH and interactions with ligands and inhibitors, as this protein provides an attractive template to further investigate the structure-function characteristics of DDAH isoforms and p/a-propeller enzymes in general. To counter the relatively large size of the enzyme for NMR studies and the difficulties to obtain unambiguous resonance assignments for the amide backbone resonances of the 58 kD homodimer, a monomeric and fully active variant of PaDDAH was engineered through rational design of site-directed mutation of two interfacial residues. The exclusively monomeric R40E- R98H double mutant protein proved more tractable by heteronuclear NMR than the wild-type homodimer. 15N backbone relaxation studies of the mutant protein, analysed in the model-free formalism, revealed that the loop that closes down on the ligand in the active site displays low values of the generalised N-H bond order parameter (S2) consistent with a high degree of mobility on the pico- to nanosecond timescale. The interaction of PaDDAH with a variety of small molecule ligands was followed by heteronuclear NMR experiments. A general observation is that ligands that act as inhibitors of the enzyme in vitro give rise to specific broadening of a subset of N-H cross peaks, including those of residues around the active site and in the loop that closes the ligand binding pocket. The results are interpreted to imply that in the bound state the dynamic profile of the backbone of the PaDDAH enzyme is altered, probably with ordering of the loop but retaining a component of conformational exchange that gives rise to residual line broadening, even in the saturated state. A similar result is obtained with a ligand covalently bound to Cys249 of the catalytic triad.