Abstract
We recently identified and characterized a novel broad substrate specificity amino acid racemase (BAR) from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3. Three genes, PH0782, PH1423, and PH1501, encoding homologs exhibiting about 45% sequence identity with BAR were present in the P. horikoshii genome. In this study, we detected pyridoxal 5′-phosphate (PLP)-dependent amino acid racemase activity in the protein encoded by PH0782. The enzyme showed activity toward Ala, Ser, Thr, and Val, but the catalytic efficiency with Thr or Val was much lower than with Ala or Ser. The enzyme was therefore designated Ala/Ser racemase (ASR). Like BAR, ASR was highly stable at high temperatures and over a wide range of pHs, though its hexameric structure differed from the dimeric structure of BAR. No activity was detected in K291A or D234A ASR mutants. This suggests that, as in Ile 2-epimerase (ILEP) from Lactobacillus buchneri JCM1115, these residues are involved in Schiff base formation and substrate interaction, respectively. Unlike BAR, enhanced ASR activity was not detected in P. horikoshii cells cultivated in the presence of D-Ala or D-Ser. This is the first description of a PLP-dependent fold type I ASR in archaea.
Highlights
D-Amino acids play a variety of important roles in many organisms
We revealed that PH0138, which was originally annotated as a putative γ-aminobutyric acid aminotransferase (GABA-AT), catalyzed the racemization of a number of hydrophobic and aromatic amino acids
We found that three other genes, PH0782, PH1423, and PH1501, were annotated as putative GABA-AT genes and had high sequence similarity with broad substrate specificity amino acid racemase (BAR)
Summary
D-Amino acids play a variety of important roles in many organisms. It is well known that D-Ala and D-Glu are components of the peptidoglycan cell wall (Hancock, 1960; Ghuysen, 1961), as are D-Ser and D-Asp (Reynolds and Courvalin, 2005; Veiga et al, 2006). Free D-amino acids such as D-Asp, D-Ala, and D-Ser have been. Ala/Ser Racemase From P. horikoshii detected in hyperthermophilic archaea (Matsumoto et al, 1999; Nagata et al, 1999; Long et al, 2001), the cell walls of archaea such as the Pyrococcus and Thermococcus species are S-layers, composed of hexagonally or tetragonally arranged proteins or glycoproteins and do not contain D-amino acids. The physiological function of D-amino acids in archaeal cells remains unclear
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