CLONING, EXPRESSION AND CHARACTERIZATION OF THE FERREDOXIN-NADP+ REDUCTASE FROM LEPTOSPIRA INTERROGANS

CATALANO DUPUY, D; CECCARELLI, EDUARDO A.

Pinamar, Argentina

Congreso; 10th Congress Panamerican Association for Biochemistry and Molecular Biology; 2005

SAIB - PABMB

Ferredoxin-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 how this catalytic improvement was accomplished but probably was obtained by subtle changes in the protein structure 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 parameters than the pea FNR. We have obtained protein crystals to elucidate its structure by means of x-rays crystallography and to compare it with the structures of other FNRs.