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Cloning, expression, purification, crystallization and preliminary X‐ray diffraction analysis of N ‐acetylmannosamine kinase from methicillin‐resistant Staphylococcus aureus
Author(s) -
North Rachel A.,
Seizova Simona,
Stampfli Anja,
Kessans Sarah A.,
Suzuki Hironori,
Griffin Michael D. W.,
Kvansakul Marc,
Dobson Renwick C. J.
Publication year - 2014
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x14007250
Subject(s) - biochemistry , escherichia coli , sialic acid , staphylococcus aureus , affinity chromatography , microbiology and biotechnology , biology , enzyme , chemistry , gene , bacteria , genetics
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 as Escherichia coli , Staphylococcus aureus , Vibrio cholerae and V. 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 for S. aureus growth on sialic acids, suggesting that the pathway is a viable antimicrobial drug target. The enzyme N ‐acetylmannosamine kinase is involved in the catabolism of sialic acid, transferring a phosphate group from adenosine‐5′‐triphosphate to the C6 position of N ‐acetylmannosamine to generate N ‐acetylmannosamine‐6‐phosphate. The gene was cloned into an appropriate expression vector; recombinant protein was expressed in E. coli BL21 (DE3) cells and purified via anion‐exchange chromatography, hydrophobic interaction chromatography and size‐exclusion chromatography. Purified N ‐acetylmannosamine kinase was screened for crystallization. The best crystal diffracted to a resolution of beyond 2.6 Å in space group P 2. Understanding the structural nature of this enzyme from methicillin‐resistant S. aureus will provide insights necessary for the development of future antimicrobials.