Cloning, expression and characterization of methionine adenosyltransferase in Leishmania infantum promastigotes

doi:10.1074/jbc.M105512200

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Papers In Press, published online ahead of print November 6, 2001
J. Biol. Chem, 10.1074/jbc.M105512200
Submitted on June 14, 2001
Revised on October 29, 2001
Accepted on November 5, 2001

Cloning, expression and characterization of methionine adenosyltransferase in Leishmania infantum promastigotes

Rosa M. Reguera, Rafael Balana-Fouce, Yolanda Perez-Pertejo, Francisco J. Fernandez, Carlos Garcia-Estrada, Juan C. Cubria, Cesar Ordonez, and David Ordonez

Farmacologia y Toxicologia, Universidad de Leon, Leon, Leon 24071

Corresponding Author: dftrrt{at}unileon.es

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, sulphuration 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 sigmoid behavior and is weakly inhibited by AdoMet, whereas tripolyphosphatase activity has sigmoid 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, while downstream sequences appeared to have no bearing on expression.

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