ENHANCED CRYSTALLIZATION OF Leptospira interrogans HEME OXYGENASE BY DIRECTED MUTAGENESIS

SOLDANO, A; CATALANO DUPUY, D. L.; CECCARELLI, E. A.

Rosario

Congreso; L Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2014

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

LepHO is a heme oxygenase from Leptospira interrogans, the causative agent of leptospirosis. It catalyzes the conversion of heme to iron, biliverdin and carbon monoxide employing oxygen and reducing equivalents. Efforts to crystallize this enzyme have been unsuccessful. Therefore, various modifications were designed to obtain a more compact and stable protein abolishing dimer formation and removing the C-terminal 20 amino acids. By structural modeling we have inferred that this region is exposed. Cys-26 and Glu-205 were replaced with Ser and a stop codon respectively. All LepHO mutants were cloned and expressed in Escherichia coli cells and the recombinant productswere purified as soluble proteins. Their enzymatic properties were analyzed by UV-visible spectroscopy. We confirmed that all mutants were able to bind the substrate heme and complete its turnover while receiving electrons from its redox partner, the ferredoxin-NADP+ reductase. Their activities were comparable with that of the wild-type LepHO. Crystals of heme complexes were obtained by the sitting drop vapor diffusion method using 24% (w/v) PEG 4000 and 0.2 M sodium acetate in 0.1 M Tris-HCl (pH 8.5) as precipitant. Only mutants containing a stop codon in position 205 were able to crystallize, suggesting that the C-terminal impedes the crystals formation. LepHO structure elucidation is in progress.