Cloning, expression and characterization of the Ferredoxin-NADP+ reductase from Leptospira interrogans

CATALANO DUPUY, DANIELA L.; CECCARELLI, EDUARDO A.

Pinamar, Buenos Aires, ARGENTINA

Congreso; XLI Reunión Anual de SAIB; 2005

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Ferredoxin (flavodoxin)-NADP(H) reductases (FNR) are ubiquitous flavoenzymes that deliver NADPH or low potential one-electron donors (ferredoxin, flavodoxin) to redox-based metabolisms in plastids, mitochondria and bacteria. There are differences in catalytic efficiencies among the members of the FNR family. Whereas plant FNRs display turnover numbers related to the needs of the photosynthetic process, bacterial reductases are much less active. It is not known hoow this catalytic improvement was accomplished but probably was obtained by subtle changes in the protein estructure and FAD conformation. Recently, we have determined that FNR from Leptospira interrogans (LepFNR), a parasitic bacterium of animals and humans, belong to an already defined monophyletic group composed entirely of parasitic species and it is included among the plastidic FNR class, probably as the result of a lateral gene transfer event. Therefore, we decided to study the function and structure of this enzyme. We cloned the LepFNR gene from the bacterium genome, and developed an efficient expression and purification system of the protein. We found that LepFNR displays similar spectral properties, functional characteristics and kinetic parameter than pea FNR. We have obtained protein crystal to elucidate its structure by means of x-rays crystallography and to compare it with the structures of other FNRs.