A solanesyl-diphosphate synthase localizes in glycosomes of Trypanosoma cruzi.
We report the cloning of a Trypanosoma cruzi gene encoding a solanesyl-diphosphate synthase, TcSPPS. The amino acid sequence (molecular mass approximately 39 kDa) is homologous to polyprenyl-diphosphate synthases from different organisms, showing the seven conserved motifs and the typical hydrophobic profile. TcSPPS preferred geranylgeranyl diphosphate as the allylic substrate. The final product, as determined by TLC, had nine isoprene units. This suggests that the parasite synthesizes mainly ubiquinone-9 (UQ-9), as described for Trypanosoma brucei and Leishmania major. In fact, that was the length of the ubiquinone extracted from epimastigotes, as determined by high-performance liquid chromatography. Expression of TcSPPS was able to complement an Escherichia coli ispB mutant. A punctuated pattern in the cytoplasm of the parasite was detected by immunofluorescence analysis with a specific polyclonal antibody against TcSPPS. An overlapping fluorescence pattern was observed using an antibody directed against the glycosomal marker pyruvate phosphate dikinase, suggesting that this step of the isoprenoid biosynthetic pathway is located in the glycosomes. Co-localization in glycosomes was confirmed by immunogold electron microscopy and subcellular fractionation. Because UQ has a central role in energy production and in reoxidation of reduction equivalents, TcSPPS is promising as a new chemotherapeutic target.
Ferella
Marcela
M
Instituto Nacional de Parasitología Dr. M. Fatala Chabén, Av. Paseo Colón 568, Administración Nacional de Laboratorios e Institutos de Salud, Ministerio de Salud, Buenos Aires 1063, Argentina.
Montalvetti
Andrea
A
Rohloff
Peter
P
Miranda
Kildare
K
Fang
Jianmin
J
Reina
Silvia
S
Kawamukai
Makoto
M
Búa
Jacqueline
J
Nilsson
Daniel
D
Pravia
Carlos
C
Katzin
Alejandro
A
Cassera
Maria B
MB
Aslund
Lena
L
Andersson
Björn
B
Docampo
Roberto
R
Bontempi
Esteban J
EJ
D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis* / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D003360:Cosmids; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D008830:Microbodies / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000378:metabolism*; D014451:Ubiquinone / Q000737:chemistry / Q000302:isolation & purification
D014451:Ubiquinone; D019883:Alkyl and Aryl Transferases; C022644:trans-octaprenyltranstransferase; C030571:ubiquinone 9