, Volume 22, Issue 3, pp 395–405 | Cite as

Crystal structure of the NADP+ and tartrate-bound complex of l-serine 3-dehydrogenase from the hyperthermophilic archaeon Pyrobaculum calidifontis

  • Kazunari Yoneda
  • Haruhiko Sakuraba
  • Tomohiro Araki
  • Toshihisa Ohshima
Original Paper


A gene encoding l-serine dehydrogenase (l-SerDH) that exhibits extremely low sequence identity to the Agrobacterium tumefaciens l-SerDH was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The predicted amino acid sequence showed 36% identity with that of Pseudomonas aeruginosa l-SerDH, suggesting that P. calidifontis l-SerDH is a novel type of l-SerDH, like Ps. aeruginosa l-SerDH. The overexpressed enzyme appears to be the most thermostable l-SerDH described to date, and no loss of activity was observed by incubation for 30 min at temperatures up to 100 °C. The enzyme showed substantial reactivity towards d-serine, in addition to l-serine. Two different crystal structures of P. calidifontis l-SerDH were determined using the Se-MAD and MR method: the structure in complex with NADP+/sulfate ion at 1.18 Å and the structure in complex with NADP+/l-tartrate (substrate analog) at 1.57 Å. The fold of the catalytic domain showed similarity with that of Ps. aeruginosa l-SerDH. However, the active site structure significantly differed between the two enzymes. Based on the structure of the tartrate, l- and d-serine and 3-hydroxypropionate molecules were modeled into the active site and the substrate binding modes were estimated. A structural comparison suggests that the wide cavity at the substrate binding site is likely responsible for the high reactivity of the enzyme toward both l- and d-serine enantiomers. This is the first description of the structure of the novel type of l-SerDH with bound NADP+ and substrate analog, and it provides new insight into the substrate binding mechanism of l-SerDH. The results obtained here may be very informative for the creation of l- or d-serine-specific SerDH by protein engineering.


Archaea Crystal structure l-Serine 3-dehydrogenase Hyperthermophile Pyrobaculum calidifontis strain JCM 11548/VA1 



l-Serine 3-dehydrogenase




Selenium multiple-wavelength anomalous dispersion


Molecular replacement


Root-mean-square deviation


Protein data bank



The synchrotron-radiation experiment was performed at the Photon Factory BL-1A and 5A, Tsukuba, Japan. We are grateful to the staff of the Photon Factory for assistance with data collection, which was approved by the Photon Factory Program Advisory Committee (Proposal no. 2016G502). We also thank Ms. Y. Chijiiwa at the Department of Bioscience, School of Agriculture, Tokai University, for helping us with the N-terminal amino acid sequence analysis. This work was supported by a grant for JSPS KAKENHI, Grant Number 16K18689, a grant from the Tokai University Educational System, and a grant from the Institute for Fermentation, Osaka (IFO).

Author contributions

KY performed the experiments and wrote the manuscript. KY, HS, and TO designed the study, analyzed the data, and wrote the manuscript. KY, HS, TA, and TO participated in discussions during the preparation of the manuscript. All authors had final approval of the submitted and published versions.

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interest.


  1. Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66:213–221CrossRefPubMedPubMedCentralGoogle Scholar
  2. Benner SA (1982) The stereoselectivity of alcohol dehydrogenases: a stereochemical imperative? Experientia 38:633–636CrossRefPubMedGoogle Scholar
  3. 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 72:248–254CrossRefPubMedGoogle Scholar
  4. Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL (1998) Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54:905–921CrossRefPubMedGoogle Scholar
  5. Chowdhury EK, Higuchi K, Nagata S, Misono H (1997) A novel NADP+-dependent serine dehydrogenase from Agrobacterium tumefaciens. Biosci Biotechnol Biochem 61:152–157CrossRefPubMedGoogle Scholar
  6. Doublié S (1997) Preparation of selenomethionyl proteins for phase determination. Methods Enzymol 276:523–530CrossRefGoogle Scholar
  7. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126–2132CrossRefPubMedGoogle Scholar
  8. Fujisawa H, Nagata S, Chowdhury EK, Matsumoto M, Misono H (2002) Cloning and sequencing of the serine dehydrogenase gene from Agrobacterium tumefaciens. Biosci Biotechnol Biochem 66:1137–1139CrossRefPubMedGoogle Scholar
  9. Fujisawa H, Nagata S, Misono H (2003) Characterization of short-chain dehydrogenase/reductase homologues of Escherichia coli (YdfG) and Saccharomyces cerevisiae (YMR226C). Biochim Biophys Acta 1645:89–94CrossRefPubMedGoogle Scholar
  10. Hayashi J, Inoue S, Kim K, Yoneda K, Kawarabayasi Y, Ohshima T, Sakuraba H (2015) Crystal structures of a hyperthermophilic archaeal homoserine dehydrogenase suggest a novel cofactor binding mode for oxidoreductases. Sci Rep 5:11674CrossRefPubMedPubMedCentralGoogle Scholar
  11. Kawabata T (2003) MATRAS: a program for protein 3D structure comparison. Nucleic Acids Res 31:3367–3369CrossRefPubMedPubMedCentralGoogle Scholar
  12. Kretovich VL, Stepanovich KM (1966) The enzyme catalyzing the reductive amination of oxypyruvate. Izv Akad Nauk SSSR Biol 2:295–300PubMedGoogle Scholar
  13. Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372:774–797CrossRefPubMedGoogle Scholar
  14. Lovell SC, Davis IW, Arendall WB 3rd, de Bakker PI, Word JM, Prisant MG, Richardson JS, Richardson DC (2003) Structure validation by Calpha geometry: phi, psi and Cbeta deviation. Proteins 50:437–450CrossRefPubMedGoogle Scholar
  15. Matthews BW (1968) Solvent content of protein crystals. J Mol Biol 33:491–497CrossRefPubMedGoogle Scholar
  16. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53:240–255CrossRefPubMedGoogle Scholar
  17. Ohshima T, Misono H, Soda K (1978) Properties of crystalline leucine dehydrogenase from Bacillus sphaericus. J Biol Chem 253:5719–5725PubMedGoogle Scholar
  18. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276:307–326CrossRefGoogle Scholar
  19. Tchigvintsev A, Singer A, Brown G, Flick R, Evdokimova E, Tan K, Gonzalez CF, Savchenko A, Yakunin AF (2012) Biochemical and structural studies of uncharacterized protein PA0743 from Pseudomonas aeruginosa revealed NAD+-dependent l-serine dehydrogenase. J Biol Chem 287:1874–1883CrossRefPubMedGoogle Scholar
  20. Yamazawa R, Nakajima Y, Mushiake K, Yoshimoto T, Ito K (2011) Crystal structure of serine dehydrogenase from Escherichia coli: important role of the C-terminal region for closed-complex formation. J Biochem 149:701–712CrossRefPubMedGoogle Scholar
  21. Yoneda K, Sakuraba H, Araki T, Shibata T, Nikki T, Ohshima T (2013) Crystallization and preliminary X-ray analysis of l-serine 3-dehydrogenase complexed with NADP+ from the hyperthermophilic archaeon Pyrobaculum calidifontis. Acta Crystallogr F Struct Biol Commun 69:134–136CrossRefGoogle Scholar

Copyright information

© Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  • Kazunari Yoneda
    • 1
  • Haruhiko Sakuraba
    • 2
  • Tomohiro Araki
    • 1
  • Toshihisa Ohshima
    • 3
  1. 1.Department of Bioscience, School of AgricultureTokai UniversityKumamotoJapan
  2. 2.Department of Applied Biological Science, Faculty of AgricultureKagawa UniversityKagawaJapan
  3. 3.Department of Biomedical Engineering, Faculty of EngineeringOsaka Institute of TechnologyOsakaJapan

Personalised recommendations