Human ornithine aminotransferase complexed with L-canaline and gabaculine: structural basis for substrate recognition.
BACKGROUND
Ornithine aminotransferase (OAT) is a 45 kDa pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the conversion of L-ornithine and 2-oxoglutarate to glutamate-delta-semialdehyde and glutamic acid, respectively. In humans, loss of OAT function causes an accumulation of ornithine that results in gyrate atrophy of the choroid and retina, a disease that progressively leads to blindness. In an effort to learn more about the structural basis of this enzyme's function, we have determined the X-ray structures of OAT in complex with two enzyme-activated suicide substrates: L-canaline, an ornithine analog, and gabaculine, an irreversible inhibitor of several related aminotransferases.


RESULTS
The structures of human OAT bound to the inhibitors gabaculine and L-canaline were solved to 2.3 A at 110K by difference Fourier techniques. Both inhibitors coordinate similarly in the active site, binding covalently to the PLP cofactor and causing a 20 degrees rotation in the cofactor tilt relative to the ligand-free form. Aromatic-aromatic interactions occur between the bound gabaculine molecule and active-site residues Tyr85 and Phe177, whereas Tyr55 and Arg180 provide specific contacts to the alpha-amino and carboxyl groups of L-canaline.


CONCLUSIONS
The OAT-L-canaline complex structure implicates Tyr55 and Arg180 as the residues involved in coordinating with the natural substrate ornithine during normal enzyme turnover. This correlates well with two enzyme-inactivating point mutations associated with gyrate atrophy, Tyr55-->His and Arg180-->Thr. The OAT-gabaculine complex provides the first structural evidence that the potency of the inhibitor is due to energetically favourable aromatic interactions with residues in the active site. This aromatic-binding mode may be relevant to structure-based drug design efforts against other omega-aminotransferase targets, such as GABA aminotransferase.
Shah
S A
SA
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
Shen
B W
BW
Brünger
A T
AT
D000613:Aminobutyrates / Q000737:chemistry*; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D018360:Crystallography, X-Ray; D003509:Cyclohexanecarboxylic Acids / Q000737:chemistry*; D004791:Enzyme Inhibitors; D015799:Gyrate Atrophy / Q000201:enzymology; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009953:Ornithine-Oxo-Acid Transaminase / Q000737:chemistry* / Q000235:genetics; D011732:Pyridoxal Phosphate / Q000737:chemistry; D000637:Transaminases / Q000037:antagonists & inhibitors; D014443:Tyrosine / Q000737:chemistry
D000613:Aminobutyrates; D003509:Cyclohexanecarboxylic Acids; D004791:Enzyme Inhibitors; C012987:gabaculine; D014443:Tyrosine; C001926:canaline; D011732:Pyridoxal Phosphate; D001120:Arginine; D000637:Transaminases; D009953:Ornithine-Oxo-Acid Transaminase