Crystal structure of a novel type of ornithine d-aminotransferase from the hyperthermophilic archaeon Pyrococcus horikoshii

Ornithine delta-aminotransferase (Orn-AT) activity was detected for the enzyme annotated as a gamma-aminobutyrate aminotransferase encoded by PH1423 gene from Pyrococcus horikoshii OT-3. Crystal structures of this novel archaeal omega-aminotransferase were determined for the enzyme in complex with pyridoxal 5'-phosphate (PLP), in complex with PLP and L-ornithine (L-Orn), and in complex with N-(5'-phosphopyridoxyl)-L-glutamate (PLP-L- Glu). Although the sequence identity was relatively low (28%), the main-chain coordinates of P. horikoshii OrnAT monomer showed notable similarity to those of human Orn-AT. However, the residues recognizing the alpha-amino group of L-Orn differ between the two enzymes. In human Orn-AT, Tyr55 and Tyr85 recognize the alpha-amino group, whereas the side chains of Thr92* and Asp93*, which arise from a loop in the neighboring subunit, form hydrogen bonds with the alpha-amino group of the substrate in P. horikoshii enzyme. Site-directed mutagenesis suggested that Asp93* plays critical roles in maintaining high affinity for the substrate. This study provides new insight into the substrate binding of a novel type of Orn-AT. Moreover, the structure of the enzyme with the reaction-intermediate analogue PLP-L-Glu bound provides the first structural evidence for the Glu switch mechanism in the dual substrate specificity of Orn-AT.

Automated summary

The Orn-AT activity of the enzyme encoded by the PH1423 gene from Pyrococcus horikoshii OT-3 was detected. Crystal structures of this novel archaeal omega-aminotransferase were determined in complex with pyridoxal 5'-phosphate (PLP), in complex with PLP and L-ornithine (L-Orn), and in complex with N-(5'-phosphopyridoxyl)-L-glutamate (PLP-L-Glu). The P. horikoshii OrnAT monomer showed notable similarity to that of human Orn-AT. However, the residues recognizing the alpha-amino group of L-Orn differed between the two enzymes. Asp93* was found to play a critical role in maintaining high affinity for the substrate. The structure of the enzyme with the reaction-intermediate analogue PLP-L-Glu bound provided the first structural evidence for the Glu switch mechanism in the dual substrate specificity of Orn-AT.