Residues involved in co-factor and substrate binding of the short-chain dehydrogenase/reductase PTR1 producing methotrexate resistance in Leishmania.
The Leishmania short-chain dehydrogenase/reductase gene ptr1 has been isolated while characterizing antifolate-resistant Leishmania mutants. PTR1 is active as a tetramer and can reduce pterins and folates. PTR1 has several of the hallmarks of the short chain dehydrogenase/reductase family, including a glycine rich co-factor binding site at its N-terminus, and the consensus catalytic site TyrXaa3Lys. To start probing the structure/function of PTR1, we have generated by site-directed mutagenesis five mutants, either in the co-factor-binding site, Y38D or in the catalytic site Y195F, Y195W, K199R or in a PTR1-specific region, Y175F. The mutated versions of PTR1 were studied in vivo in Leishmania and at the biochemical level using purified proteins. The Y175F mutant showed properties similar to wild-type PTR1 in every aspect tested, but all the other mutants were inactive even if they were purified as tetramers. To test the ability of the mutated PTR1 versions to bind its co-factor and substrates, trypsin digestion experiments were carried out under conditions upon which binding will prevent wild-type PTR1 of being digested by trypsin. The wild-type PTR1 as well as Y175F and Y195F mutants were protected against trypsin digestion whereas Y38D and K199R mutants were not. Mutations in regions involved in co-factor binding (Y38D) or in catalytic site (K199R) altered the binding of the ligands, explaining why those protein versions are inactive.
Leblanc
E
E
Centre de Recherche en Infectiologie du Centre de Recherche du CHUL et Département de Microbiologie, Faculté de Médecine, Université Laval, Ste-Foy, Québec, Canada.
Papadopoulou
B
B
Bernatchez
C
C
Ouellette
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003067:Coenzymes / Q000378:metabolism; D017124:Conserved Sequence; D004351:Drug Resistance; D007700:Kinetics; D007891:Leishmania / Q000187:drug effects / Q000201:enzymology*; D008727:Methotrexate / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D014162:Transfection; D014357:Trypsin
D003067:Coenzymes; D011994:Recombinant Proteins; D010088:Oxidoreductases; C085592:pteridine reductase; D014357:Trypsin; D008727:Methotrexate