HETEROLOGOUS EXPRESSION AND KINETIC ANALYSIS OF RECOMBINANT FUMARATE HYDRATASE FROM LEISHMANIA MAJOR

FQ7

Patrícia Rosa Feliciano

Maria Cristina Nonato 

Departamento de Física e Química

Faculdade de Ciências Farmacêuticas de Ribeirão Preto

Universidade de São Paulo

Ribeirão Preto 

Leishmania major Friedlin (LmjF) is a protozoan parasite responsible for Leishmaniasis that currently threatens 350 million men, women and children in 88 countries spread around the world and whose genomic sequence has been recently elucidated. In the present work, we have cloned, overexpressed, purified and kinetically analyzed the product of the gene from LmjF chromosome 24: LmjF24.0320, which encodes a protein with putative fumarate hydratase activity (LmFH-1). Fumarate hydratase, also called fumarase, catalyses the stereospecific reversible hydration of fumarate to malate. Recent studies in trypanosomatids, utilizing Trypanosoma brucei as model, suggest that fumarases are essential for survival of these parasites. LmFH-1 has been cloned in pET28a vector using L. major genomic DNA as a template.  The predicted enzyme from L. major was overexpressed in Escherichia coli strain BL21(DE3) as a histidine-tag fusion protein. Cells were grown in Luria-Bertani (LB) medium supplemented with kanamicin (30 μg/ml) and protein expression was induced by IPTG (0,25mM). LmFH-1 was purified to homogeneity by affinity chromatography in Ni-NTA (QIAGEN) column using a step gradient of imidazole. The final product was homogeneous in SDS-PAGE gel electrophoresis. Preliminary kinetic studies are performed by monitoring the product formation through spectrophotometric analysis at 250 nm. Our studies have demonstrated that LmFH-1 is readily oxidized by oxygen leading to the loss of protein activity.  For this reason, all steps, from purification to enzymatic assays, have to be performed in anaerobic conditions. Kinetic experiments are now in progress in order to determine the catalytic constants. (This work was supported by FAPESP).