Roles of conserved basic amino acid residues and activation mechanism of the hyperthermophilic aspartate racemase at high temperature.

X-ray crystallography has revealed two similar alpha/beta domains of the aspartate racemase from the hyperthermophilic archaeon, Pyrococcus horikoshii OT3. The active site is located in the cleft between the two domains where two cysteine residues face each other. This arrangement allows the substrate to enter the cleft and enables the two cysteine residues to act synergistically. However, the distance between their thiolates was estimated to be 9.6 angstroms, which is beyond the distance for cooperative action of them. We examined the molecular mechanism for the racemization reaction of this hyperthermophilic aspartate racemase by mutational analyses and molecular dynamics simulations. The mutational analyses revealed that Arg48 and Lys164 were essential for catalysis in addition to the putative catalytic cysteine residues. The molecular dynamics simulations revealed that the distance between the two active gamma-sulfur atoms of cysteine residues oscillate to periodically become shorter than the predicted cooperative distance at high temperature. In addition, the conformation of Tyr160, which is located at the entrance of the cleft and inhibits the entry of a substrate, changes periodically to open the entrance at 375 K. The opening of the gate is likely to be induced by the motion of the adjacent amino acid, Lys164. The entrance of an aspartate molecule was observed by molecular dynamics (MD) simulations driven by the force of the electrostatic interaction with Arg48, Lys164, and also Asp47. These results provide insights into the roles of amino acid residues at the catalytic site and also the activation mechanism of a hyperthermophilic aspartate racemase at high temperature.

Structural insight into gene duplication, gene fusion and domain swapping in the evolution of PLP-independent amino acid racemases.

The X-ray crystal structure has revealed two similar alpha/beta domains of aspartate racemase (AspR) from Pyrococcus horikoshii OT3, and identified a pseudo mirror-symmetric distribution of the residues around its active site [Liu et al. (2002) J. Mol. Biol. 319, 479-489]. Structural homology and functional similarity between the two domains suggested that this enzyme evolved from an ancestral domain by gene duplication and gene fusion. We have expressed solely the C-terminal domain of this AspR and determined its three-dimensional structure by X-ray crystallography. The high structural stability of this domain supports the existence of the ancestral domain. In comparison with other amino acid racemases (AARs), we suggest that gene duplication and gene fusion are conventional ways in the evolution of pyridoxal 5'-phosphate-independent AARs.

Crystal structure of aspartate racemase from Pyrococcus horikoshii OT3 and its implications for molecular mechanism of PLP-independent racemization.

There exists a d-enantiomer of aspartic acid in lactic acid bacteria and several hyperthermophilic archaea, which is biosynthesized from the l-enantiomer by aspartate racemase. Aspartate racemase is a representative pyridoxal 5'-phosphate (PLP)-independent amino acid racemase. The "two-base" catalytic mechanism has been proposed for this type of racemase, in which a pair of cysteine residues are utilized as the conjugated catalytic acid and base. We have determined the three-dimensional structure of aspartate racemase from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 at 1.9 A resolution by X-ray crystallography and refined it to a crystallographic R factor of 19.4% (R(free) of 22.2%). This is the first structure reported for aspartate racemase, indeed for any amino acid racemase from archaea. The crystal structure revealed that this enzyme forms a stable dimeric structure with a strong three-layered inter-subunit interaction, and that its subunit consists of two structurally homologous alpha/beta domains, each containing a four-stranded parallel beta-sheet flanked by six alpha-helices. Two strictly conserved cysteine residues (Cys82 and Cys194), which have been shown biochemically to act as catalytic acid and base, are located on both sides of a cleft between the two domains. The spatial arrangement of these two cysteine residues supports the "two-base" mechanism but disproves the previous hypothesis that the active site of aspartate racemase is located at the dimeric interface. The structure revealed a unique pseudo mirror-symmetry in the spatial arrangement of the residues around the active site, which may explain the molecular recognition mechanism of the mirror-symmetric aspartate enantiomers by the non-mirror-symmetric aspartate racemase.