title: Roles of conserved basic amino acid residues and activation mechanism of the hyperthermophilic aspartate racemase at high temperature.
abstract: 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.
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan.
authors.forename: Takumitsu
authors.initials: T
authors.lastname: Yoshida
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Seko
authors.forename: Okimasa
authors.initials: O
authors.lastname: Okada
authors.forename: Kousuke
authors.initials: K
authors.lastname: Iwata
authors.forename: Lijun
authors.initials: L
authors.lastname: Liu
authors.forename: Kunio
authors.initials: K
authors.lastname: Miki
authors.forename: Masafumi
authors.initials: M
authors.lastname: Yohda
mesh_terms: D000591:Amino Acid Isomerases / Q000737:chemistry*; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D043902:Pyrococcus horikoshii / Q000201:enzymology; D055672:Static Electricity; D013696:Temperature
chemical_list: D001120:Arginine; D000591:Amino Acid Isomerases; C019749:aspartate racemase; D008239:Lysine; D003545:Cysteine