Enhanced crystallization of Leptospira interrogans heme oxygenase by directed mutagenesis

SOLDANO, ANABEL; CATALANO DUPUY, DANIELA L; CECCARELLI, EDUARDO A

Rosario

Congreso; L Reunión Anual de la 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 theconversion of heme to iron, biliverdin and carbon monoxide employing oxygen and reducing equivalents. Efforts tocrystallize this enzyme have been unsuccessful. Therefore, various modifications were designed to obtain a morecompact and stable protein abolishing dimer formation and removing the C-terminal 20 amino acids. By structuralmodeling we have inferred that this region is exposed. Cys-26 and Glu-205 were replaced with Ser and a stop codonrespectively. All LepHO mutants were cloned and expressed in Escherichia coli cells and the recombinant products were purified as soluble proteins. Their enzymatic properties were analyzed by UV-visible spectroscopy. Weconfirmed that all mutants were able to bind the substrate heme and complete its turnover while receiving electronsfrom its redox partner, the ferredoxin-NADP+reductase. Their activities were comparable with that of the wild-typeLepHO. Crystals of heme complexes were obtained by the sitting drop vapor diffusion method using 24% (w/v) PEG4000 and 0.2 M sodium acetate in 0.1 M Tris-HCl (pH 8.5) as precipitant. Only mutants containing a stop codon inposition 205 were able to crystallize, suggesting that the C-terminal impedes the crystals formation. LepHO structureelucidation is in progress.