Cloning, expression, purification, and characterization of Leishmania major dihydroorotate dehydrogenase.
Leishmania major Friedlin (LmjF) is a protozoan parasite whose genomic sequence has been recently elucidated. Here we have cloned, overexpressed, purified, and characterized the product of the gene from LmjF chromosome 16: LmjF16.0530, which encodes a protein with putative dihydroorotate dehydrogenase activity. Dihydroorotate dehydrogenase (DHODH) is a flavoprotein that catalyses the oxidation of L-dihydroorotate to orotate, the fourth sequential step in the de novo pyrimidine nucleotide synthesis pathway. The predicted enzyme from L. major was cloned and expressed in Escherichia coli strain BL21(DE3) as a histidine-tag fusion protein and purified to homogeneity using affinity chromatography. The final product was homogeneous in SDS-PAGE gel electrophoresis. The dihydroorotate oxidase activity has been assayed and the steady-state kinetic mechanism has been determined using fumarate as the oxidizing substrate. The catalysis by LmDHODH enzyme proceeds by a Ping-Pong Bi-Bi mechanism and the kinetic parameters Km were calculated to be 90 and 418 microM for dihydroorotate and fumarate, respectively, and Vmax was calculated to be 11 micromol min-1 mg-1. Our results confirmed that the product of the gene LmjF16.0530, whose function has previously been predicted based on homology to known proteins, can therefore be positively assigned as L. major DHODH.
Feliciano
Patrícia R
PR
Laboratório de Cristalografia de Proteínas, Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. Café S/N Ribeirão Preto, S.P. 14040-903, Brazil.
Cordeiro
Artur T
AT
Costa-Filho
Antonio J
AJ
Nonato
M Cristina
MC
D000818:Animals; D002384:Catalysis; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D000090722:Dihydroorotate Dehydrogenase; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D018320:Leishmania major / Q000201:enzymology* / Q000235:genetics; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D000090722:Dihydroorotate Dehydrogenase; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D044925:Oxidoreductases Acting on CH-CH Group Donors