Leishmania donovani methionine adenosyltransferase. Role of cysteine residues in the recombinant enzyme.
Methionine adenosyltransferase (MAT, EC 2.5.1.6)-mediated synthesis of S-adenosylmethionine (AdoMet) is a two-step process consisting of the formation of AdoMet and the subsequent cleavage of the tripolyphosphate (PPPi) molecule, a reaction induced, in turn, by AdoMet. The fact that the two activities, AdoMet synthesis and tripolyphosphate hydrolysis, can be measured separately is particularly useful when the site-directed mutagenesis approach is used to determine the functional role of the amino acid residues involved in each. The present report describes the cloning and subsequent functional refolding, using a bacterial expression system, of the MAT gene (GenBank accession number AF179714) from Leishmania donovani, the etiological agent of visceral leishmaniasis. The absolute need to include a sulfhydryl-protection reagent in the refolding buffer for this protein, in conjunction with the rapid inactivation of the functionally refolded protein by N-ethylmaleimide, suggests the presence of crucial cysteine residues in the primary structure of the MAT protein. The seven cysteines in L. donovani MAT were mutated to their isosterical amino acid, serine. The C22S, C44S, C92S and C305S mutants showed a drastic loss of AdoMet synthesis activity compared to the wild type, and the C33S and C47S mutants retained a mere 12% of wild-type MAT activity. C106S mutant activity and kinetics remained unchanged with respect to the wild-type. Cysteine substitutions also modified PPPi cleavage and AdoMet induction. The C22S, C44S and C305S mutants lacked in tripolyphosphatase activity altogether, whereas C33S, C47S and C92S retained low but detectable activity. The behavior of the C92S mutant was notable: its inability to synthesize AdoMet combined with its retention of tripolyphosphatase activity appear to be indicative of the specific involvement of the respective residue in the first step of the MAT reaction.
Pérez-Pertejo
Yolanda
Y
Departamento de Farmacología y Toxicología (INTOXCAL), Universidad de León, León, Spain.
Reguera
Rosa M
RM
Villa
Hector
H
García-Estrada
Carlos
C
Balaña-Fouce
Rafael
R
Pajares
Maria A
MA
Ordóñez
David
D
D019943:Amino Acid Substitution; D000818:Animals; D003001:Cloning, Molecular; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D025461:Feedback, Physiological; D006868:Hydrolysis; D007893:Leishmania donovani / Q000201:enzymology*; D008716:Methionine Adenosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011122:Polyphosphates / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism
D011122:Polyphosphates; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D008716:Methionine Adenosyltransferase; D003545:Cysteine; C005692:triphosphoric acid