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Cloning, expression, purification, crystallization and preliminary X‐ray diffraction analysis of N ‐acetylmannosamine‐6‐phosphate 2‐epimerase from methicillin‐resistant Staphylococcus aureus
Author(s) -
North Rachel A.,
Kessans Sarah A.,
Griffin Michael D. W.,
Watson Andrew J. A.,
Fairbanks Antony J.,
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/s2053230x14007171
Subject(s) - biochemistry , escherichia coli , sialic acid , enzyme , microbiology and biotechnology , biology , gene cluster , staphylococcus aureus , catabolism , cloning (programming) , gene , bacteria , chemistry , genetics , computer science , programming language
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 P 2 1 2 1 2. 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.