Cloning expression and characterization of methionine adenosyltransferase in Leishmania infantum promastigotes.
Methionine adenosyltransferase (MAT) catalyzes the synthesis of s-adenosylmethionine (AdoMet), a metabolite that plays an important role in a variety of cellular functions, such as methylation, sulfuration, and polyamine synthesis. In this study, genomic DNA from the protozoan parasite Leishmania infantum was cloned and characterized. L. infantum MAT, unlike mammalian MAT, is codified by two identical genes in a tandem arrangement and is only weakly regulated by AdoMet. L. infantum MAT mRNA is expressed as a single transcript, with the enzyme forming a homodimer with tripolyphosphatase in addition to MAT activity. Expression of L. infantum MAT in Escherichia coli proves that the MAT and tripolyphosphatase activities are functional in vivo. MAT shows sigmoidal behavior and is weakly inhibited by AdoMet, whereas tripolyphosphatase activity has sigmoidal behavior and is strongly activated by AdoMet. Plasmids containing the regions flanking MAT2 were fused immediately upstream and downstream of the luciferase-coding region and transfected into L. infantum. Subsequent examination of luciferase activity showed that homologous expression in L. infantum promastigotes was dramatically dependent on the presence of polypyrimidine tracts and a spliced leader junction site upstream of the luciferase gene, whereas downstream sequences appeared to have no bearing on expression.
Reguera
Rosa M
RM
Departamento de Farmacologia y Toxicologia, Facultad de Veterinaria, Universidad de León Campus de Vegazana s/n, 24071 León, Spain. dftrrt@isidoro.unileon.es
Balaña-Fouce
Rafael
R
Pérez-Pertejo
Yolanda
Y
Fernández
Francisco J
FJ
García-Estrada
Carlos
C
Cubría
Juan C
JC
Ordóñez
César
C
Ordóñez
David
D
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D007700:Kinetics; D018314:Leishmania infantum / Q000201:enzymology*; D008156:Luciferases / Q000235:genetics; D008716:Methionine Adenosyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012436:S-Adenosylmethionine / Q000096:biosynthesis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014162:Transfection
D017931:DNA Primers; D016054:DNA, Protozoan; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D008156:Luciferases; D008716:Methionine Adenosyltransferase