title: Fumarate hydratase isoforms of Leishmania major: subcellular localization, structural and kinetic properties.
abstract: Fumarate hydratases (FHs; EC 4.2.1.2) are enzymes that catalyze the reversible hydration of fumarate to S-malate. Parasitic protists that belong to the genus Leishmania and are responsible for a complex of vector-borne diseases named leishmaniases possess two genes that encode distinct putative FH enzymes. Genome sequence analysis of Leishmania major Friedlin reveals the existence of genes LmjF24.0320 and LmjF29.1960 encoding the putative enzymes LmFH-1 and LmFH-2, respectively. In the present work, the FH activity of both L. major enzymes has been confirmed. Circular dichroism studies suggest important differences in terms of secondary structure content when comparing LmFH isoforms and even larger differences when comparing them to the homologous human enzyme. CD melting experiments revealed that both LmFH isoforms are thermolabile enzymes. The catalytic efficiency under aerobic and anaerobic environments suggests that they are both highly sensitive to oxidation and damaged by oxygen. Intracellular localization studies located LmFH-1 in the mitochondrion, whereas LmFH-2 was found predominantly in the cytosol with possibly also some in glycosomes. The high degree of sequence conservation in different Leishmania species, together with the relevance of FH activity for the energy metabolism in these parasites suggest that FHs might be exploited as targets for broad-spectrum antileishmanial drugs.
authors.affiliation: Laboratório de Cristalografia de Proteínas, FCFRP, Universidade de São Paulo, Ribeirão Preto 14040-903, Brazil.
authors.forename: Patrícia R
authors.initials: PR
authors.lastname: Feliciano
authors.forename: Shreedhara
authors.initials: S
authors.lastname: Gupta
authors.forename: Fabio
authors.initials: F
authors.lastname: Dyszy
authors.forename: Marcelo
authors.initials: M
authors.lastname: Dias-Baruffi
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Costa-Filho
authors.forename: Paul A M
authors.initials: PA
authors.lastname: Michels
authors.forename: M Cristina
authors.initials: MC
authors.lastname: Nonato
mesh_terms: D002942:Circular Dichroism; D003001:Cloning, Molecular; D005649:Fumarate Hydratase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016680:Genome, Bacterial; D007527:Isoenzymes; D007700:Kinetics; D018320:Leishmania major / Q000201:enzymology* / Q000235:genetics; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D005649:Fumarate Hydratase