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 14C-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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aswad DW (1984) Determination of d- and l-aspartate in amino acid mixtures by high-performance liquid chromatography after derivatization with a chiral adduct of o-phthaldialdehyde. Anal Biochem 137:405–409
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Cohen GN, Barbe V, Flament D, Galperin M, Heilig R, Lecompte O, Poch O, Prieur D, Querellou J, Ripp R, Thierry JC, Van der Oost J, Weissenbach J, Zivanovic Y, Forterre P (2003) An integrated analysis of the genome of the hyperthermophilic archaeon Pyrococcus abyssi. Mol Microbiol 47:1495–1512
Gonzalez JM, Masuchi Y, Robb FT, Ammerman JW, Maeder DL, Yanagibayashi M, Tamaoka J, Kato C (1998) Pyrococcus horikoshii sp. nov., a hyperthermophilic archaeon isolated from a hydrothermal vent at the Okinawa Trough. Extremophiles 2:123–130
Grishin NV, Phillips MA, Goldsmith EJ (1995) Modeling of the spatial structure of eukaryotic ornithine decarboxylases. Protein Sci 4:1291–1304
Hashimoto A, Nishikawa T, Oka T, Takahashi K, Hayashi T (1992) Determination of free amino acid enantiomers in rat brain and serum by high-performance liquid chromatography after derivatization with N-tert.-butyloxycarbonyl-l-cysteine and o-phthaldialdehyde. J Chromatogr 582:41–48
Hoaki T, Wirsen CO, Hanzawa S, Maruyama T, Jannasch HW (1993) Amino acid requirements of two hyperthermophilic archaeal isolates from deep-sea vents, Desulfurococcus strain SY and Pyrococcus strain GB-D. Appl Environ Microbiol 59:610–613
Hoaki T, Nishijima M, Kato M, Adachi K, Mizobuchi S, Hanzawa N, Maruyama T (1994) Growth requirements of hyperthermophilic sulfur-dependent heterotrophic archaea isolated from a shallow submarine geothermal system with reference to their essential amino acids. Appl Environ Microbiol 60:2898–2904
Kawakami R, Sakuraba H, Kamohara S, Goda S, Kawarabayasi Y, Ohshima T (2004) Oxidative stress response in an anaerobic hyperthermophilic archaeon: presence of a functional peroxiredoxin in Pyrococcus horikoshii. J Biochem 136:541–547
Kawakami R, Sakuraba H, Tsuge H, Goda S, Katunuma N, Ohshima T (2005) A second novel dye-linked l-proline dehydrogenase complex is present in the hyperthermophilic archaeon Pyrococcus horikoshii OT-3. FEBS J 272:4044–4054
Kawarabayasi Y, Sawada M, Horikawa H, Haikawa Y, Hino Y, Yamamoto S, Sekine M, Baba S, Kosugi H, Hosoyama A, Nagai Y, Sakai M, Ogura K, Otsuka R, Nakazawa H, Takamiya M, Ohfuku Y, Funahashi T, Tanaka T, Kudoh Y, Yamazaki J, Kushida N, Oguchi A, Aoki K, Kikuchi H (1998) Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3. DNA Res 5:55–76
Kita A, Tasaki S, Yohda M, Miki K (2009) Crystal structure of PH1733, an aspartate racemase homologue, from Pyrococcus horikoshii OT3. Proteins 74:240–244
Knaggs AR (2003) The biosynthesis of shikimate metabolites. Nat Prod Rep 20:119–136
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Liu L, Iwata K, Kita A, Kawarabayasi Y, Yohda M, Miki K (2002) Crystal structure of aspartate racemase from Pyrococcus horikoshii OT3 and its implications for molecular mechanism of PLP-independent racemization. J Mol Biol 319:479–489
Long Z, Lee JA, Okamoto T, Sekine M, Nimura N, Imai K, Yohda M, Maruyama T, Sumi M, Kamo N, Yamagishi A, Oshima T, Homma H (2001) Occurrence of d-amino acids and a pyridoxal 5′-phosphate-dependent aspartate racemase in the acidothermophilic archaeon, Thermoplasma acidophilum. Biochem Biophys Res Commun 281:317–321
Matsumoto M, Homma H, Long Z, Imai K, Iida T, Maruyama T, Aikawa Y, Endo I, Yohda M (1999) Occurrence of free d-amino acids and aspartate racemases in hyperthermophilic archaea. J Bacteriol 181:6560–6563
Mutaguchi Y, Ohmori T, Wakamatsu T, Doi K, Ohshima T (2013) Identification, purification, and characterization of a novel amino acid racemase, isoleucine 2-epimerase, from Lactobacillus species. J Bacteriol 195:5207–5215
Nagata Y, Tanaka K, Iida T, Kera Y, Yamada R, Nakajima Y, Fujiwara T, Fukumori Y, Yamanaka T, Koga Y, Tsuji S, Kawaguchi-Nagata K (1999) Occurrence of d-amino acids in a few archaea and dehydrogenase activities in hyperthermophile Pyrobaculum islandicum. Biochim Biophys Acta 1435:160–166
Ohnishi M, Saito M, Wakabayashi S, Ishizuka M, Nishimura K, Nagata Y, Kasai S (2008) Purification and characterization of serine racemase from a hyperthermophilic archaeon, Pyrobaculum islandicum. J Bacteriol 190:1359–1365
Ohtaki A, Nakano Y, Iizuka R, Arakawa T, Yamada K, Odaka M, Yohda M (2008) Structure of aspartate racemase complexed with a dual substrate analogue, citric acid, and implications for the reaction mechanism. Proteins 70:1167–1174
Robb FT, Place AR (1995) Media for Thermophiles. In: Robb FT, Place AR (eds) Archaea-a laboratory manual-thermophiles. Cold Spring Harbor Laboratory Press, New York, pp 167–168
Sakuraba H, Satomura T, Kawakami R, Yamamoto S, Kawarabayasi Y, Kikuchi H, Ohshima T (2002) l-Aspartate oxidase is present in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3: characteristics and role in the de novo biosynthesis of nicotinamide adenine dinucleotide proposed by genome sequencing. Extremophiles 6:275–281
Tishkov VI, Khoronenkova SV (2005) d-Amino acid oxidase: structure, catalytic mechanism, and practical application. Biochemistry (Mosc) 70:40–54
Watrin L, Martin-Jezequel V, Prieur D (1995) Minimal amino acid requirements of the hyperthermophilic archaeon Pyrococcus abyssi, isolated from deep-sea hydrothermal vents. Appl Environ Microbiol 61:1138–1140
Yoshida T, Seko T, Okada O, Iwata K, Liu L, Miki K, Yohda M (2006) Roles of conserved basic amino acid residues and activation mechanism of the hyperthermophilic aspartate racemase at high temperature. Proteins 64:502–512
Yoshimura T, Esaki N (2003) Amino acid racemases: functions and mechanisms. J Biosci Bioeng 96:103–109
Acknowledgments
We are grateful to Mr. Yuji Kawahara and Ms. Kumi Yamashita for their excellent technical assistance.
Conflict of interest
The authors declare that they have no conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Additional information
Handling Editor: J. Broos.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Kawakami, R., Ohmori, T., Sakuraba, H. et al. Identification of a novel amino acid racemase from a hyperthermophilic archaeon Pyrococcus horikoshii OT-3 induced by d-amino acids. Amino Acids 47, 1579–1587 (2015). https://doi.org/10.1007/s00726-015-2001-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00726-015-2001-6