Identification of a novel amino acid racemase from a hyperthermophilic archaeon Pyrococcus horikoshii OT-3 induced by d-amino acids

Abstract

To date, there have been few reports analyzing the amino acid requirement for growth of hyperthermophilic archaea. We here found that the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 requires Thr, Leu, Val, Phe, Tyr, Trp, His and Arg in the medium for growth, and shows slow growth in medium lacking Met or Ile. This largely corresponds to the presence, or absence, of genes related to amino acid biosynthesis in its genome, though there are exceptions. The amino acid requirements were dramatically lost by addition of D-isomers of Met, Leu, Val, allo-Ile, Phe, Tyr, Trp and Arg. Tracer analysis using (14)C-labeled D-Trp showed that D-Trp in the medium was used as a protein component in the cells, suggesting the presence of D-amino acid metabolic enzymes. Pyridoxal 5'-phosphate (PLP)-dependent racemase activity toward Met, Leu and Phe was detected in crude extract of P. horikoshii and was enhanced in cells grown in the medium supplemented with D-amino acids, especially D-allo-Ile. The gene encoding the racemase was narrowed down to one open reading frame on the basis of enzyme purification from P. horikoshii cells, and the recombinant enzyme exhibited PLP-dependent racemase activity toward several amino acids, including Met, Leu and Phe, but not Pro, Asp or Glu. This is the first report showing the presence in a hyperthermophilic archaeon of a PLP-dependent amino acid racemase with broad substrate specificity that is likely responsible for utilization of D-amino acids for growth.

Distinctive Roles of D-Amino Acids in the Homochiral World: Chirality of Amino Acids Modulates Mammalian Physiology and Pathology

“…However, there are several reports of the occurrence of d-amino acids in archaea, such as d-Asp in some hyperthermophilic archaea 26,27 and in Thermoplasma acidophilum, 28 and d-Ser in Py- robaculum islandicum, 29 although their functions are not well understood. A more recent report states that Pyrococcus horikoshii, a hyperthermophilic archeon, metabolizes several d-amino acids with a broad-spectrum racemase for their growth, 30 suggesting that a group of archaea fully utilizes d-amino acids as nutrients obtained in the extreme environments in which they live.…”

A Novel PLP-Dependent Alanine/Serine Racemase From the Hyperthermophilic Archaeon Pyrococcus horikoshii OT-3

“…The thermostability of PH0782 was assessed by determining the residual activity after incubation at selected temperatures for 30 min and at 80°C for several different incubation times. The residual activities in the temperature and pH stability assays were determined spectrophotometrically using L -Ala as the substrate, as described previously ( Kawakami et al, 2015 ).…”

“…The activity of BAR was greatly increased in P. horikoshii cells grown on medium supplemented with D - allo -Ile ( Kawakami et al, 2015 ). We therefore investigated ASR activity in P. horikoshii cells grown on medium supplemented with D -Ala, D -Ser or D - allo -Ile.…”

“…This includes detailed structural and functional characterization of a PLP-independent AspR from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 ( Liu et al, 2001 , 2002a , b ; Yoshida et al, 2006 ; Ohtaki et al, 2008 ; Kita et al, 2009 ). We recently detected novel PLP-dependent amino acid racemase activity toward Met, Leu, and Phe in the crude extract of P. horikoshii , identified the enzyme gene (ORF ID: PH0138 ), and determined the properties of the recombinant protein ( Kawakami et al, 2015 , 2017 ). That enzyme showed activity toward 10 amino acids and was therefore named BAR.…”

“…This suggests these homologs also function as amino acid racemases. We previously reported that BAR activity is markedly increased in P. horikoshii cells grown on medium supplemented with D - allo -Ile, which suggests BAR mediates utilization of D -amino acids for growth ( Kawakami et al, 2015 ). In the present study, we constructed expression systems for BAR homologs using pET vector and detected amino acid racemase activity toward Ala and Ser in the recombinant PH0782 enzyme.…”