Identification of pantoate kinase and phosphopantothenate synthetase from Methanospirillum hungatei

J Biosci Bioeng. 2013 Apr;115(4):372-6. doi: 10.1016/j.jbiosc.2012.10.019. Epub 2012 Nov 27.

Abstract

Pantothenate synthetase (PanC) and pantothenate kinase which function in the canonical coenzyme A (CoA) biosynthetic pathway cannot be found in most archaea. COG1829 and COG1701 intrinsic to archaea were proposed as the candidate proteins for producing 4'-phosphopantothenate instead, and the COG1701 protein from Methanosarcina mazei was assigned as PanC. Meanwhile, the Thermococcus kodakarensis COG1829 and COG1701 proteins were biochemically identified as novel enzymes, i.e., pantoate kinase (PoK) and phosphopantothenate synthetase (PPS). In this study, the functions of Mhun_0831 (COG1829) and Mhun_0832 (COG1701) from Methanospirillum hungatei were identified, and the recombinant enzymes were partially characterized. Plasmids simultaneously possessing the two genes encoding Mhun_0831 and Mhun_0832 complemented the poor growth of the temperature-sensitive Escherichia coli pantothenate kinase mutant ts9. The recombinant Mhun_0831 and Mhun_0832 expressed in E. coli cells exhibited PoK and PPS activities, respectively, being in accord with the functions of T. kodakarensis proteins. The PoK activity was most active at pH 8.5 and 40°C, and accepted ATP and UTP as a phosphate donor. Although CoA did not affect the PoK activity, the end product considerably accelerated the PPS activity. The homologs of both proteins are widely conserved in most archaeal genomes. Taken together, our findings indicate that archaea can synthesize CoA through the unique pathway involving PoK and PPS, in addition to the canonical one that the order Thermoplasmatales employs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Coenzyme A / metabolism
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Ligases / genetics
  • Ligases / metabolism*
  • Methanospirillum / enzymology*
  • Methanospirillum / genetics
  • Pantothenic Acid / analogs & derivatives*
  • Pantothenic Acid / biosynthesis
  • Phosphotransferases (Alcohol Group Acceptor) / genetics
  • Phosphotransferases / genetics
  • Phosphotransferases / metabolism*
  • Uridine Triphosphate / metabolism

Substances

  • Pantothenic Acid
  • phosphopantothenic acid
  • Phosphotransferases
  • Phosphotransferases (Alcohol Group Acceptor)
  • pantothenate kinase
  • Ligases
  • Coenzyme A
  • Uridine Triphosphate