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Extremophiles

, Volume 20, Issue 4, pp 385–393 | Cite as

Structural and functional characterization of aspartate racemase from the acidothermophilic archaeon Picrophilus torridus

  • Takayuki Aihara
  • Toshiya Ito
  • Yasuaki Yamanaka
  • Keiichi Noguchi
  • Masafumi Odaka
  • Masae Sekine
  • Hiroshi Homma
  • Masafumi Yohda
Original Paper

Abstract

Functional and structural characterizations of pyridoxal 5′-phosphate-independent aspartate racemase of the acidothermophilic archaeon Picrophilus torridus were performed. Picrophilus aspartate racemase exhibited high substrate specificity to aspartic acid. The optimal reaction temperature was 60 °C, which is almost the same as the optimal growth temperature. Reflecting the low pH in the cytosol, the optimal reaction pH of Picrophilus aspartate racemase was approximately 5.5. However, the activity at the putative cytosolic pH of 4.6 was approximately 6 times lower than that at the optimal pH of 5.5. The crystal structure of Picrophilus aspartate racemase was almost the same as that of other pyridoxal 5′-phosphate -independent aspartate racemases. In two molecules of the dimer, one molecule contained a tartaric acid molecule in the catalytic site; the structure of the other molecule was relatively flexible. Finally, we examined the intracellular existence of d-amino acids. Unexpectedly, the proportion of d-aspartate to total aspartate was not very high. In contrast, both d-proline and d-alanine were observed. Because Picrophilus aspartate racemase is highly specific to aspartate, other amino acid racemases might exist in Picrophilus torridus.

Keywords

Aspartate racemase Picrophilus torridus Archaea d-Amino acid Crystal structure 

Abbreviations

PtoAspR

Aspartate racemase of Picrophilus torridus

TFA

Trifluoroacetic acid

NBD-F

4-Fluoro-7-nitrobenzofurazan

PLP

Pyridoxal 5′-phosphate

Notes

Acknowledgments

We are grateful to the beamline assistants at the Photon Factory (PF) for data collection at the beamlines BL-5A, BL-6A and AR-NW12.

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Copyright information

© Springer Japan 2016

Authors and Affiliations

  • Takayuki Aihara
    • 1
  • Toshiya Ito
    • 1
  • Yasuaki Yamanaka
    • 1
  • Keiichi Noguchi
    • 2
  • Masafumi Odaka
    • 3
  • Masae Sekine
    • 4
  • Hiroshi Homma
    • 4
  • Masafumi Yohda
    • 1
  1. 1.Department of Biotechnology and Life Science, Graduate School of TechnologyTokyo University of Agriculture and TechnologyKoganeiJapan
  2. 2.Instrumentation Analysis CenterTokyo University of Agriculture and TechnologyKoganeiJapan
  3. 3.Department of Life Science, Faculty and Graduate School of Engineering and Resource ScienceAkita UniversityAkita CityJapan
  4. 4.Department of Pharmaceutical and Life SciencesKitasato UniversityTokyoJapan

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