Mutagenesis study on the role of a lysine residue highly conserved in formate dehydrogenases and periplasmic nitrate reductases

Biochem Biophys Res Commun. 2003 Oct 10;310(1):40-7. doi: 10.1016/j.bbrc.2003.08.114.

Abstract

Lysine 85 (K85) in the primary structure of the catalytic subunit of the periplasmic nitrate reductase (NAP-A) of Ralstonia eutropha H16 is highly conserved in periplasmic nitrate reductases and in the structurally related catalytic subunit of the formate dehydrogenases of various bacterial species. It is located between an [4Fe-4S] center and one of the molybdopterin-guanine dinucleotides mediating the through bonds electron flow to convert the specific substrate of the respective enzymes. To examine the role of K85, the structure of NAP-A of R. eutropha strain H16 was modeled on the basis of the crystal structure from the Desulfovibrio desulfuricans enzyme (Dias et al. Structure Fold Des. 7(1) (1999) 65) and K85 was replaced by site-directed mutagenesis, yielding K85R and K85M, respectively. The specific nitrate reductase activity was determined in periplasmic extracts. The mutant enzyme carrying K85R showed 23% of the wild-type activity, whereas the replacement by a polar, uncharged residue (K85M) resulted in complete loss of the catalytic activity. The reduced nitrate reductase activity of K85R was not due to different quantities of the expressed gene product, as controlled immunologically by NAP-specific antibodies. The results indicate that K85 is optimized for the electron transport flux to reduce nitrate to nitrite in NAP-A, and that the positive charge alone cannot meet further structural requirement for efficient electron flow.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cupriavidus necator / enzymology
  • Formate Dehydrogenases / chemistry
  • Formate Dehydrogenases / metabolism*
  • Lysine / metabolism*
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis
  • Nitrate Reductase
  • Nitrate Reductases / chemistry
  • Nitrate Reductases / metabolism*
  • Periplasm / enzymology*
  • Sequence Homology, Amino Acid

Substances

  • Formate Dehydrogenases
  • Nitrate Reductases
  • Nitrate Reductase
  • Lysine