Crystal structure of human ornithine aminotransferase complexed with the highly specific and potent inhibitor 5-fluoromethylornithine

Ornithine aminotransferase (L-ornithine:2-oxoacid delta-aminotransferase; E C 2.6.1.13), a pyridoxal-5'-phosphate-dependent mitochondrial enzyme contro ls the L-ornithine level in tissues by catalyzing the transfer of the delta -amino group of L-ornithine to 2-oxoglutarate, producing L-glutamate-gamma- semialdehyde and L-glutamate. (2S,5S)-5-Fluoromethylornithine is the only i nhibitor exclusively specific for ornithine aminotransferase known to date. Both in vitro and in vivo, it blocks the enzyme by a suicide reaction lead ing to a covalent adduct with the cofactor. The crystal structure of the en zyme-inhibitor complex was solved at a resolution of 1.95 Angstrom. No sign ificant conformational changes compared with the native enzyme structure we re observed. The structure reveals the atomic details of the cofactor-inhib itor adduct and its interactions with the active site of the enzyme. The ma in residues responsible for specific binding of the inhibitor are Arg180, w hich forms a strong salt bridge with the alpha-carboxylate and Tyr55, which is involved in a short hydrogen bond with the ol-amino group. The experime ntal observation that in the racemic mixture, (2S,5S)-5-fluoromethylornithi ne is exclusively responsible for the enzyme inhibition can be explained on the basis of the active site topology. Model building studies strongly sug gest that the natural substrate L-ornithine, in its external aldimine adduc t with the enzyme, makes use of the same recognition site as the inhibitor. It is proposed that the neutralization of the active site Arg413 by a salt bridge with Glu235 also plays an important role in productive binding of b oth 5-fluoromethylornithine and L-ornithine. Arg180 and Arg413 are believed to be instrumental in recognition of L-glutamate, by binding its gamma and alpha-carboxylate groups, respectively. This requires a different side-cha in conformation of Glu235. Lys292 is the only obvious candidate for catalyz ing the rate-limiting proton transfer steps in the transamination reaction. (C) 1999 Academic Press.