Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of N-acetylmannosamine-6-phosphate 2-epimerase from methicillin-resistant Staphylococcus aureus

Acta Crystallogr F Struct Biol Commun. 2014 May;70(Pt 5):650-5. doi: 10.1107/S2053230X14007171. Epub 2014 Apr 17.

Abstract

Sialic acids are one of the most important carbohydrate classes in biology. Some bacterial pathogens can scavenge sialic acids from their surrounding environment and degrade them as a source of carbon, nitrogen and energy. This sequestration and subsequent catabolism of sialic acid require a cluster of genes known as the `Nan-Nag' cluster. The enzymes coded by these genes are important for pathogen colonization and persistence. Importantly, the Nan-Nag genes have proven to be essential for Staphylococcus aureus growth on sialic acids, suggesting that the pathway is a viable antibiotic drug target. The enzyme N-acetylmannosamine-6-phosphate 2-epimerase is involved in the catabolism of sialic acid; specifically, the enzyme converts N-acetylmannosamine-6-phosphate into N-acetylglucosamine-6-phosphate. The gene was cloned into an appropriate expression vector, and recombinant protein was expressed in Escherichia coli BL21 (DE3) cells and purified via a three-step procedure. Purified N-acetylmannosamine-6-phosphate 2-epimerase was screened for crystallization. The best crystal diffracted to a resolution of beyond 1.84 Å in space group P21212. Understanding the structural nature of this enzyme from methicillin-resistant S. aureus will provide us with the insights necessary for the development of future antibiotics.

Keywords: MRSA; N-acetylmannosamine-6-phosphate 2-epimerase; NanE; Staphylococcus aureus; sialic acid catabolism.

Publication types

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

MeSH terms

  • Bacterial Proteins / biosynthesis*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Carbohydrate Epimerases / biosynthesis*
  • Carbohydrate Epimerases / genetics
  • Carbohydrate Epimerases / isolation & purification
  • Cloning, Molecular* / methods
  • Crystallization
  • Gene Expression Regulation, Enzymologic*
  • Methicillin-Resistant Staphylococcus aureus / enzymology*
  • X-Ray Diffraction

Substances

  • Bacterial Proteins
  • Carbohydrate Epimerases
  • N-acetylmannosamine-6-phosphate epimerase