Cloning and expression of a lombricine kinase from an echiuroid worm: insights into structural correlates of substrate specificity

Biochem Biophys Res Commun. 2002 Mar 8;291(4):939-44. doi: 10.1006/bbrc.2002.6539.

Abstract

Phosphagen kinases constitute a large family of enzymes catalyzing the reversible phosphorylation of guanidino acceptor compounds. These guanidino substrates differ substantially in size and chemical properties. In spite of the appearance of X-ray crystal structures for two members of this family, creatine kinase (CK) and arginine kinase (AK), the structural correlates of substrate specificity remain to be fully elucidated. We have determined the cDNA and deduced amino acid sequences for lombricine (guanidinethylphosphoserine) kinase (LK) from the echiuroid worm Urechis caupo and expressed the cDNA in Escherichia coli. The recombinant protein was purified by affinity chromatography and showed high capacity for phosphorylation of lombricine. Phosphagen kinases consist of a small, N-terminal domain and a much larger domain connected by a linker sequence. A key event in catalysis in CK and AK, and certainly all other phosphagen kinases, is a large conformational change involving involving a rotation of the two domains and the movement of two highly conserved flexible loops (one located in the small domain; the other located in the large domain of these enzymes) which clamp down on the substrates. Multiple sequence alignments of Urechis LK with the only other LK sequence available and CK, AK and glycocyamine kinase sequences, confirm the importance of the small flexible loop located in the N-terminal domain of phosphagen kinases as one component of the structural determinants of guanidine specificity. The role of the other flexible loop in the large domain in terms of substrate specificity remains questionable.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Annelida / enzymology
  • Chickens
  • Cloning, Molecular
  • Consensus Sequence
  • Escherichia coli / genetics
  • Invertebrates / enzymology*
  • Molecular Sequence Data
  • Phosphotransferases (Nitrogenous Group Acceptor) / chemistry
  • Phosphotransferases (Nitrogenous Group Acceptor) / genetics*
  • Phosphotransferases (Nitrogenous Group Acceptor) / metabolism*
  • Protein Structure, Tertiary
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Serine / analogs & derivatives*
  • Serine / chemistry
  • Serine / metabolism
  • Substrate Specificity

Substances

  • Recombinant Proteins
  • Serine
  • lombricine
  • Phosphotransferases (Nitrogenous Group Acceptor)
  • lombricine kinase