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Cloning, expression, and biochemical characterization of 3-deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase from the hyperthermophilic bacterium Aquifex pyrophilus

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Abstract

3-Deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase, catalyzes the aldol-type condensation between phosphoenolpyruvate (PEP) and d-arabinose-5-phosphate (A5P) to produce the unusual 8-carbon sugar KDO8P, and inorganic phosphate. A 15.5-kb segment containing the kdsA gene from the hyperthermophilic bacterium Aquifex pyrophilus was cloned from a genomic library and sequenced. The native kdsA gene lacks a typical ribosome binding site, but contains a conserved U,A-rich sequence upstream to the start codon. The purified kdsA gene product catalyzes the formation of KDO8P from its natural substrates, PEP and A5P, as determined by 1H NMR analysis. KDO8P synthase showed maximum activity at 80 °C and pH 5.5–6.0 at 10-min reaction assay. At temperatures of 70, 80, and 90 °C, the enzyme exhibited half-lives of 8.0, 2.25, and 0.5 h, respectively. The kinetic constants at 60 °C were K m A5P=70 μM, K m PEP=290 μM, and k cat=4 s−1. The isolated enzyme contained 0.19 and 0.26 mol iron and zinc, respectively, per mole of enzyme subunit. Treatment with metal chelators eliminated enzyme activity, and by the addition of several divalent metal ions, the activity was restored and even exceeded the original activity. These results indicate that A. pyrophilus KDO8P synthase is a metal-dependent enzyme. A C11A mutant of KDO8P synthase from A. pyrophulis retained less than 1% of the wild-type activity and was shown to be incapable of metal binding.

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Acknowledgments

We thank Prof. Gyu Yu (Korea Institute of Science and Technology, Korea) for providing the genomic library of A. pyrophilus, and Prof. Karl O. Stetter (University of Regensburg, Germany) for providing the chromosomal DNA of A. pyrophilus. This work was supported by the Rubin Scientific and Medical Fund for promotion of research at the Technion (grant number 060-624), and by U.S.–Israel Binational Science Foundation Grant 97-356 (T.B.). Additional support was provided by the Fund for the Promotion of Research at the Technion, and by the Otto Meyerhof Center for Biotechnology, Technion, established by the Minerva Foundation (Munich, Germany).

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Authors and Affiliations

  1. Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, 32000 , Haifa, Israel

    Smadar Shulami & Yuval Shoham

  2. Department of Chemistry, Haifa, Israel

    Smadar Shulami, Emilia Rabkin & Timor Baasov

  3. The Interdepartmental Program in Biotechnology, Haifa, Israel

    Orit Yaniv

  4. Institute of Catalysis Science and Technology, Haifa, Israel

    Yuval Shoham & Timor Baasov

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  1. Smadar Shulami
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  2. Orit Yaniv
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Correspondence to Timor Baasov.

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Communicated by G. Antranikian

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Shulami, S., Yaniv, O., Rabkin, E. et al. Cloning, expression, and biochemical characterization of 3-deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase from the hyperthermophilic bacterium Aquifex pyrophilus . Extremophiles 7, 471–481 (2003). https://doi.org/10.1007/s00792-003-0346-3

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