The molecular structure of hyperthermostable aromatic aminotransferase with novel substrate specificity from Pyrococcus horikoshii

Aromatic amino acid aminotransferase (ArATPh), which has a melting temperat ure of 120 degrees C, is one of the most thermostable aminotransferases yet to be discovered. The crystal structure of this aminotransferase from the hyperthermophilic archaeon Pyrococcus horikoshii was determined to a resolu tion of 2.1 Angstrom ArATPh has a homodimer structure in which each subunit is composed of two domains, in a manner similar to other well characterize d aminotransferases, By the least square fit after superposing on a mesophi lic ArAT, the ArATPh molecule exhibits a large deviation of the main chain coordinates, three shortened alpha-helices, an elongated loop connecting tw o domains, and a long loop transformed from an alpha-helix, which are all f actors that are likely to contribute to its hyperthermostability, The pyrid ine ring of the cofactor pyridoxal 5'-phosphate covalently binding to Lys(2 33) is stacked parallel to F121 on one side and interacts with the geminal dimethyl-CH/pi groups of Val(201) on the other side. This tight stacking ag ainst the pyridine ring probably contributes to the hyperthermostability of ArATPh, Compared with other ArATs, ArATPh has a novel substrate specificit y, the order of preference being Tyr > Phe > Glu > Trp > His much greater t han Met > Leu > Asp > Asn. Its relatively weak activity against Asp is due to lack of an arginine residue corresponding to Arg(292)* (where the asteri sk indicates that this is a residues supplied by the other subunit of the d imer) in pig cytosolic aspartate aminotransferase. The enzyme recognizes th e aromatic substrate by hydrophobic interaction with aromatic rings (Phe(12 1) and Tyr(59)*) and probably recognizes acidic substrates by a hydrophilic interaction involving a hydrogen bond network with Thr(264)*.