Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization.
Two different putative galactokinase genes, found in the genome database of Trypanosoma cruzi were cloned and sequenced. Expression of the genes in Escherichia coli resulted for TcGALK-1 in the synthesis of a soluble and active enzyme, and in the case of TcGALK-2 gene a less soluble protein, with predicted molecular masses of 51.9kDa and 51.3kDa, respectively. The Km values determined for the recombinant proteins were for galactose 0.108mM (TcGALK-1) and 0.091mM (TcGALK-2) and for ATP 0.36mM (TcGALK-1) and 0.1mM (TcGALK-2). Substrate inhibition by ATP (Ki 0.414mM) was only observed for TcGALK-2. Gel-filtration chromatography showed that natural TcGALKs and recombinant TcGALK-1 are monomeric. In agreement with the possession of a type-1 peroxisome-targeting signal by both TcGALKs, they were found to be present inside glycosomes using two different methods of subcellular fractionation in conjunction with mass spectrometry. Both genes are expressed in epimastigote and trypomastigote stages since the respective proteins were immunodetected by western blotting. The T. cruzi galactokinases present their highest (52-47%) sequence identity with their counterpart from Leishmania spp., followed by prokaryotic galactokinases such as those from E. coli and Lactococcus lactis (26-23%). In a phylogenetic analysis, the trypanosomatid galactokinases form a separate cluster, showing an affiliation with bacteria. Epimastigotes of T. cruzi can grow in glucose-depleted LIT-medium supplemented with 20mM of galactose, suggesting that this hexose, upon phosphorylation by a TcGALK, could be used in the synthesis of UDP-galactose and also as a possible carbon and energy source.
Laboratorio de Enzimología de Parásitos, Facultad de Ciencias, Universidad de Los Andes, La Hechicera, 5101 Mérida, Venezuela.
Ángel E
ÁE
Lobo-Rojas
Laboratorio de Enzimología de Parásitos, Facultad de Ciencias, Universidad de Los Andes, La Hechicera, 5101 Mérida, Venezuela.
Eglys B
EB
González-Marcano
Laboratorio de Enzimología de Parásitos, Facultad de Ciencias, Universidad de Los Andes, La Hechicera, 5101 Mérida, Venezuela.
Edward A
EA
Valera-Vera
Laboratorio de Enzimología de Parásitos, Facultad de Ciencias, Universidad de Los Andes, La Hechicera, 5101 Mérida, Venezuela.
Héctor R
HR
Acosta
Laboratorio de Enzimología de Parásitos, Facultad de Ciencias, Universidad de Los Andes, La Hechicera, 5101 Mérida, Venezuela.
Wilfredo A
WA
Quiñones
Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, Scotland.
Richard J S
RJ
Burchmore
Laboratorio de Enzimología de Parásitos, Facultad de Ciencias, Universidad de Los Andes, La Hechicera, 5101 Mérida, Venezuela.
Juan L
JL
Concepción
Laboratorio de Enzimología de Parásitos, Facultad de Ciencias, Universidad de Los Andes, La Hechicera, 5101 Mérida, Venezuela. Electronic address: acaceres@ula.ve.
Ana J
AJ
Cáceres
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005686:Galactokinase / Q000235:genetics*; D005690:Galactose / Q000378:metabolism*; D008830:Microbodies / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014349:Trypanosoma cruzi / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*
D011994:Recombinant Proteins; D005686:Galactokinase; D005690:Galactose
GHMP superfamily; Galactokinase; Galactose metabolism; Glycosome; Kinetic analysis; Nucleotide sugars; Trypanosoma cruzi; UDP-galactose