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

Abstract

Aromatic amino acid aminotransferase (ArATPh), which has a melting temperature of 120 °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 resolution of 2.1 Å ArATPh has a homodimer structure in which each subunit is composed of two domains, in a manner similar to other well characterized aminotransferases. By the least square fit after superposing on a mesophilic ArAT, the ArATPh molecule exhibits a large deviation of the main chain coordinates, three shortened α-helices, an elongated loop connecting two domains, and a long loop transformed from an α-helix, which are all factors that are likely to contribute to its hyperthermostability. The pyridine ring of the cofactor pyridoxal 5'-phosphate covalently binding to Lys233 is stacked parallel to F121 on one side and interacts with the germinal dimethyl-CH/π groups of Val201 on the other side. This tight stacking against the pyridine ring probably contributes to the hyperthermostability of ArATPh. Compared with other ArATs, ArATPh has a novel substrate specificity, the order of preference being Tyr > Phe > Glu > Trp > His >> Met > Leu > Asp >Asn. Its relatively weak activity against Asp is due to lack of an arginine residue corresponding to Arg292* (where the asterisk indicates that this is a residues supplied by the other subunit of the dimer) in pig cytosolic aspartate aminotransferase. The enzyme recognizes the aromatic substrate by hydrophobic interaction with aromatic rings (Phe121 and Tyr59*) and probably recognizes acidic substrates by a hydrophilic interaction involving a hydrogen bond network with Thr264*.