title: Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of N-acetylmannosamine-6-phosphate 2-epimerase from methicillin-resistant Staphylococcus aureus.
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.
authors.affiliation: Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8041, New Zealand.
authors.forename: Rachel A
authors.initials: RA
authors.lastname: North
authors.affiliation: Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8041, New Zealand.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Kessans
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010, Australia.
authors.forename: Michael D W
authors.initials: MD
authors.lastname: Griffin
authors.affiliation: Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch 8041, New Zealand.
authors.forename: Andrew J A
authors.initials: AJ
authors.lastname: Watson
authors.affiliation: Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch 8041, New Zealand.
authors.forename: Antony J
authors.initials: AJ
authors.lastname: Fairbanks
authors.affiliation: Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8041, New Zealand.
authors.forename: Renwick C J
authors.initials: RC
authors.lastname: Dobson
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002238:Carbohydrate Epimerases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular / Q000379:methods; D003460:Crystallization; D015971:Gene Expression Regulation, Enzymologic; D055624:Methicillin-Resistant Staphylococcus aureus / Q000201:enzymology*; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D002238:Carbohydrate Epimerases; C120657:N-acetylmannosamine-6-phosphate epimerase
keywords: MRSA; N-acetylmannosamine-6-phosphate 2-epimerase; NanE; Staphylococcus aureus; sialic acid catabolism