Bulletin of the Korean Chemical Society

Korean Chemical Society (대한화학회)
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Discovery and Characterization of a Thermostable NADH Oxidase from Pyrococcus horikoshii OT3

  • Koh, Jong-Uk (Department of Chemistry, College of Natural Sciences, Chung-Ang University) ;
  • Chung, Hyun-Jung (Department of Chemistry, College of Natural Sciences, Chung-Ang University) ;
  • Chang, Woo-Young (Department of Chemistry, College of Natural Sciences, Chung-Ang University) ;
  • Tanokura, Masaru (Graduate School of Agricultural and Life Sciences, The University of Tokyo) ;
  • Kong, Kwang-Hoon (Department of Chemistry, College of Natural Sciences, Chung-Ang University)
  • Published : 2009.12.20
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Abstract

A gene (PH0311) encoding a hypothetical protein from the genome sequence data of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 was cloned and over-expressed in Escherichia coli. The purified recombinant protein was found to possess FAD-dependent NADH oxidase activity, although it lacked sequence homology to any other known general NADH oxidase family. The product of the PH0311 gene was thus designated PhNOX (NADH oxidase from Pyrococcus horikoshii), with an estimated molecular weight of 84 kDa by gel filtration and 22 kDa by SDS-PAGE, indicating it to be a homotetramer of 22 kDa subunits. PhNOX catalyzed the oxidation of reduced ${\beta}$-NADH with subsequent formation of $H_2O_2$ in the presence of FAD as a cofactor, but not ${\alpha}$-NADH, ${\alpha}$-NADPH, or ${\beta}$-NADPH. PhNOX showed high affinity for ${\beta}$-NADH with a Km value of 3.70 ${\mu}$M and exhibited optimum activity at pH 8.0 and 95$^{\circ}C$ as it is highly stable against high temperature.

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