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Roles of conserved basic amino acid residues and activation mechanism of the hyperthermophilic aspartate racemase at high temperature
Author(s) -
Yoshida Takumitsu,
Seko Tomohiro,
Okada Okimasa,
Iwata Kousuke,
Liu Lijun,
Miki Kunio,
Yohda Masafumi
Publication year - 2006
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.21010
Subject(s) - pyrococcus horikoshii , cysteine , molecular dynamics , amino acid , racemization , stereochemistry , chemistry , aspartic acid , active site , residue (chemistry) , crystallography , biophysics , biochemistry , catalysis , biology , enzyme , computational chemistry
X‐ray crystallography has revealed two similar α/β 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 Å, 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 γ‐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. Proteins 2006. © 2006 Wiley‐Liss, Inc.