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

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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.

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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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Authors and Affiliations

  1. Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, 615-8510, Japan

    Takuya Ishibashi, Hiroya Tomita, Yuusuke Yokooji & Haruyuki Atomi

  2. Institute for Chemical Research, Kyoto University, Gokasho, Uji, 611-0011, Japan

    Tatsuya Morikita, Bunta Watanabe & Jun Hiratake

  3. Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto, 606-8502, Japan

    Asako Kishimoto & Kunio Miki

  4. Research Reactor Institute, Kyoto University, Kumatori, Sennan, Osaka, 590-0494, Japan

    Akiko Kita

  5. Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Noji-Higashi, Kusatsu, Shiga, 525-8577, Japan

    Tadayuki Imanaka

  6. JST, CREST, Sanbancho, Chiyoda-ku, Tokyo, 102-0075, Japan

    Kunio Miki, Tadayuki Imanaka & Haruyuki Atomi

Authors
  1. Takuya Ishibashi
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  2. Hiroya Tomita
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  3. Yuusuke Yokooji
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  4. Tatsuya Morikita
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  5. Bunta Watanabe
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  6. Jun Hiratake
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  7. Asako Kishimoto
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  8. Akiko Kita
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  9. Kunio Miki
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  10. Tadayuki Imanaka
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  11. Haruyuki Atomi
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Corresponding author

Correspondence to Haruyuki Atomi.

Additional information

Communicated by A. Driessen.

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Ishibashi, T., Tomita, H., Yokooji, Y. et al. A detailed biochemical characterization of phosphopantothenate synthetase, a novel enzyme involved in coenzyme A biosynthesis in the Archaea. Extremophiles 16, 819–828 (2012). https://doi.org/10.1007/s00792-012-0477-5

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  • DOI: https://doi.org/10.1007/s00792-012-0477-5

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