Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of N-acetylmannosamine kinase from methicillin-resistant Staphylococcus aureus Academic Article uri icon

abstract

  • N-Acetylmannosamine kinase (EC 2.7.1.60) is involved in the catabolism of sialic acid for many bacterial pathogens implicated in human disease such asEscherichia coli,Staphylococcus aureus,Vibrio choleraeandV. vulnificus. Interestingly, some human commensals and bacterial pathogens can scavenge sialic acids from their surrounding environment and degrade them as a source of carbon, nitrogen and energy. This process requires a cluster of genes known as the `Nan-Nag cluster', which have proven to be essential forS. aureusgrowth on sialic acids, suggesting that the pathway is a viable antimicrobial drug target. The enzymeN-acetylmannosamine kinase is involved in the catabolism of sialic acid, transferring a phosphate group from adenosine-5′-triphosphate to the C6 position ofN-acetylmannosamine to generateN-acetylmannosamine-6-phosphate. The gene was cloned into an appropriate expression vector; recombinant protein was expressed inE. coliBL21 (DE3) cells and purifiedviaanion-exchange chromatography, hydrophobic interaction chromatography and size-exclusion chromatography. PurifiedN-acetylmannosamine kinase was screened for crystallization. The best crystal diffracted to a resolution of beyond 2.6 Å in space groupP2. Understanding the structural nature of this enzyme from methicillin-resistantS. aureuswill provide insights necessary for the development of future antimicrobials.

authors

  • North, RA
  • Seizova, S
  • Stampfli, A
  • Kessans, SA
  • Suzuki, H
  • Griffin, MDW
  • Kvansakul, M
  • Dobson, RCJ

publication date

  • 2014