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

• Published in: Journal of molecular biology 1999-01, Vol.285 (1), p.297-309
• Main Authors: Storici, P, Capitani, G, Müller, R, Schirmer, T, Jansonius, J N
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Binding Sites; Crystallography, X-Ray; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - metabolism; Glutamic Acid; Humans; Microspectrophotometry; Models, Molecular; Molecular Sequence Data; Molecular Structure; Ornithine - analogs & derivatives; Ornithine - chemistry; Ornithine - metabolism; Ornithine-Oxo-Acid Transaminase - antagonists & inhibitors; Ornithine-Oxo-Acid Transaminase - chemistry; Ornithine-Oxo-Acid Transaminase - metabolism; Protein Conformation; Solutions; Substrate Specificity
• ISSN: 0022-2836
• DOI: 10.1006/jmbi.1998.2289

Ornithine aminotransferase (l-ornithine:2-oxoacid delta-aminotransferase; EC 2.6.1.13), a pyridoxal-5'-phosphate-dependent mitochondrial enzyme controls 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 inhibitor exclusively specific for ornithine aminotransferase known to date. Both in vitro and in vivo, it blocks the enzyme by a suicide reaction leading to a covalent adduct with the cofactor. The crystal structure of the enzyme-inhibitor complex was solved at a resolution of 1.95 A. No significant conformational changes compared with the native enzyme structure were observed. The structure reveals the atomic details of the cofactor-inhibitor adduct and its interactions with the active site of the enzyme. The main residues responsible for specific binding of the inhibitor are Arg180, which forms a strong salt bridge with the alpha-carboxylate and Tyr55, which is involved in a short hydrogen bond with the alpha-amino group. The experimental observation that in the racemic mixture, (2S, 5S)-5-fluoromethylornithine is exclusively responsible for the enzyme inhibition can be explained on the basis of the active site topology. Model building studies strongly suggest that the natural substrate l-ornithine, in its external aldimine adduct 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 both 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-chain conformation of Glu235. Lys292 is the only obvious candidate for catalyzing the rate-limiting proton transfer steps in the transamination reaction.