Mutational analysis of methionine adenosyltransferase from Leishmania donovani.
The 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. This report describes the mutational analysis of the amino acids involved in both the ATP and L-methionine binding sites of Leishmania donovani MAT (GenBank accession number AF179714) the aetiological agent of visceral leishmaniasis. Site-directed mutagenesis was used to substitute neutral residues for the basic amino acid (Lys168, Lys256, Lys276, Lys280 and His17), acidic residues (Asp19, Asp121, Asp166, Asp249, Asp277 and Asp288) and Phe241 involved in AdoMet synthesis and PPPi hydrolysis. With the exception of D116N, none of these mutants was able to synthesize AdoMet at a significant rate, although H17A, H17N, K256A, K280A, D19N, D121N, D166N, D249N and D282N showed measurable tripolyphosphatase activity. Finally, the C-terminus domain of L. donovani MAT was truncated at three points (F382Stop, D375Stop, F368Stop), deleting a 3(10) one-turn helix motif in all three cases. Whilst none of the truncated proteins conserved MAT activity, they were able to hydrolyse PPPi, albeit at a lower rate than the wild-type enzyme. A fourth protein with an internal deletion (E376DeltaF382) in the C-terminal domain conserved high tripolyphosphatase activity, which was not, however, induced by 50 microM AdoMet.
Pérez-Pertejo
Yolanda
Y
Departamento de Farmacología y Toxicología (INTOXCAL), Universidad de León, Spain.
Reguera
Rosa M
RM
García-Estrada
Carlos
C
Balaña-Fouce
Rafael
R
Ordóñez
David
D
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D007893:Leishmania donovani / Q000201:enzymology*; D008716:Methionine Adenosyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D017931:DNA Primers; D011994:Recombinant Proteins; D008716:Methionine Adenosyltransferase