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Extremophiles

, Volume 16, Issue 6, pp 819–828 | Cite as

A detailed biochemical characterization of phosphopantothenate synthetase, a novel enzyme involved in coenzyme A biosynthesis in the Archaea

  • Takuya Ishibashi
  • Hiroya Tomita
  • Yuusuke Yokooji
  • Tatsuya Morikita
  • Bunta Watanabe
  • Jun Hiratake
  • Asako Kishimoto
  • Akiko Kita
  • Kunio Miki
  • Tadayuki Imanaka
  • Haruyuki Atomi
Original Paper

Abstract

We have previously reported that the majority of the archaea utilize a novel pathway for coenzyme A biosynthesis (CoA). Bacteria/eukaryotes commonly use pantothenate synthetase and pantothenate kinase to convert pantoate to 4′-phosphopantothenate. However, in the hyperthermophilic archaeon Thermococcus kodakarensis, two novel enzymes specific to the archaea, pantoate kinase and phosphopantothenate synthetase, are responsible for this conversion. Here, we examined the enzymatic properties of the archaeal phosphopantothenate synthetase, which catalyzes the ATP-dependent condensation of 4-phosphopantoate and β-alanine. The activation energy of the phosphopantothenate synthetase reaction was 82.3 kJ mol−1. In terms of substrate specificity toward nucleoside triphosphates, the enzyme displayed a strict preference for ATP. Among several amine substrates, activity was detected with β-alanine, but not with γ-aminobutyrate, glycine nor aspartate. The phosphopantothenate synthetase reaction followed Michaelis–Menten kinetics toward β-alanine, whereas substrate inhibition was observed with 4-phosphopantoate and ATP. Feedback inhibition by CoA/acetyl-CoA and product inhibition by 4′-phosphopantothenate were not observed. By contrast, the other archaeal enzyme pantoate kinase displayed product inhibition by 4-phosphopantoate in a non-competitive manner. Based on our results, we discuss the regulation of CoA biosynthesis in the archaea.

Keywords

Coenzyme A Phosphopantothenate synthetase Pantoate kinase Thermococcus Archaea 

Notes

Acknowledgments

This study was partly carried out with the NMR and MS spectrometers in the Joint Usage/Research Center (JURC) at Institute for Chemical Research, Kyoto University.

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Copyright information

© Springer 2012

Authors and Affiliations

  • Takuya Ishibashi
    • 1
  • Hiroya Tomita
    • 1
  • Yuusuke Yokooji
    • 1
  • Tatsuya Morikita
    • 2
  • Bunta Watanabe
    • 2
  • Jun Hiratake
    • 2
  • Asako Kishimoto
    • 3
  • Akiko Kita
    • 4
  • Kunio Miki
    • 3
    • 6
  • Tadayuki Imanaka
    • 5
    • 6
  • Haruyuki Atomi
    • 1
    • 6
  1. 1.Department of Synthetic Chemistry and Biological Chemistry, Graduate School of EngineeringKyoto UniversityKyotoJapan
  2. 2.Institute for Chemical ResearchKyoto UniversityUjiJapan
  3. 3.Department of Chemistry, Graduate School of ScienceKyoto UniversityKyotoJapan
  4. 4.Research Reactor InstituteKyoto UniversitySennanJapan
  5. 5.Department of Biotechnology, College of Life SciencesRitsumeikan UniversityKusatsuJapan
  6. 6.JST, CRESTTokyoJapan

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