Structural studies on dihydrouridine synthase A (DusA) from Pseudomonas aeruginosa.

ABSTRACT

Dihydrouridination is one of the abundant modifications in tRNA editing. The presence of dihydrouridine is attributed to tRNA stability desired for the efficient gene translation process. The conversion of uridine to dihydrouridine is catalyzed by flavine containing enzyme called dihydrouridine synthase (Dus). We report first ever information about DusA enzyme from Pseudomonas aeruginosa in form of structural and functional studies. The gene coding for DusA from P. aeruginosa (PADusA) was cloned, expressed and purified, using recombinant DNA technology methods. Thermal and chemical stability of PADusA was determined with respect to temperature and urea-induced equilibrium unfolding experiments, with monitoring the change of ellipticity at 200-260 nm by Circular Dichroism (CD) spectroscopy. Unfolding studies revealed that PADusA has acquired a stable tertiary structure fold with a Tm value of 46.2 °C and Cm of 2.7 M for urea. The enzyme contains 43% α-helices and 16% ß-strands. The three dimensional structure of PADusA was modeled using insilico methods. In order to understand the mechanism of substrate recognition and catalysis, tRNA and puromycin were docked on PADusA structure and their binding was analyzed. The structural features suggested that PADusA may also form a novel target for structure based drug design of antimicrobial agents.