N-Acetyl-L-glutamate kinase from Escherichia coli: cloning of the gene, purification and crystallization of the recombinant enzyme and preliminary X-ray analysis of the free and ligand-bound forms

Acta Crystallogr D Biol Crystallogr. 1999 Jul;55(Pt 7):1350-2. doi: 10.1107/s0907444999005351.

Abstract

The gene for Escherichia coli N-acetyl-L-glutamate kinase (NAGK) was cloned in a plasmid and expressed in E. coli, allowing enzyme purification in three steps. NAGK exhibits high specific activity (1.1 micromol s-1 mg-1), lacks Met1 and forms dimers (shown by cross-linking). Crystals of unliganded NAGK diffract to 2 A and belong to space group P6122 or its enantiomorph P6522 (unit-cell parameters a = b = 78.6, c = 278.0 A) with two monomers in the asymmetric unit. Crystals of NAGK with acetylglutamate and the ATP analogue AMPPNP diffract to 1.8 A and belong to space group C2221 (unit-cell parameters a = 60.0, b = 71.9, c = 107.4 A), with one monomer in the asymmetric unit. NAGK crystallization will allow the determination of proposed structural similarities to carbamate kinase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cloning, Molecular
  • Crystallization
  • Crystallography, X-Ray
  • DNA Primers
  • Escherichia coli / enzymology*
  • Phosphotransferases (Carboxyl Group Acceptor) / chemistry*
  • Phosphotransferases (Carboxyl Group Acceptor) / genetics
  • Phosphotransferases (Carboxyl Group Acceptor) / isolation & purification
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification

Substances

  • DNA Primers
  • Recombinant Proteins
  • Phosphotransferases (Carboxyl Group Acceptor)
  • acetylglutamate kinase