Crystal structures of two different 4-aminobutyrate aminotransferase-like racemases from the hyperthermophilic...

A broad substrate-specificity amino acid racemase (BAR; PH0138 gene product) exhibiting the activity toward ten amino acids and an alanine/serine-specific racemase (ASR; PH0782 gene product) were identified in the hyperthermophilic archaeon Pyrococcus horikoshii OT-3. Both enzymes were originally annotated as 4-aminobutyrate aminotransferase (GABA-AT) but were phylogenetically distinct from conventional GABA-ATs. In this study, we analyzed and compared the crystal structures of BAR and ASR. Crystal structures of BAR were determined in complex with N-(5'-phosphopyridoxyl)-l-isoleucine (PLP-l-Ile), N-(5'-phosphopyridoxyl)-d-allo-isoleucine (PLP-d-allo-Ile), and N-(5'-phosphopyridoxyl)-d-phenylalanine (PLP-d-Phe). On the other hand, crystal structures of ASR were determined in complex with N-(5'-phosphopyridoxyl)-l-alanine (PLP-l-Ala) and N-(5'-phosphopyridoxyl)-d-alanine (PLP-d-Ala). Structural comparisons of BAR revealed that the Phe35 and Trp463 side chains moved depending on the substrate, suggesting that the conformational flexibility of these side chains contributes to the broad substrate specificity of BAR. A structural comparison of BAR with ASR revealed that Trp456, Leu317*, and Met89* in ASR (the asterisk indicates a residue in the adjacent subunit) could sterically interfere with the side chain of d- or l-Phe, the preferred substrate of BAR. However, none of the ASR mutants tested (W456A, W456F, W456L, L317V, and M89T) showed detectable activity toward l-Phe. On the other hand, the L317V mutant of ASR exhibited altered substrate specificity toward linear amino acids, increasing the preference for l-2-aminobutyrate over l-Ala. In addition, substitutions at Trp456 in ASR severely reduced the activity toward l-Ala, suggesting that this residue is important for maintaining a productive substrate-binding geometry in ASR.