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Allosteric Regulation of an Anthrax Enzyme

The non-hydrolysing bacterial UDP-GlcNAc 2-epimerases catalyse the reversible conversion of UDP-N-acetylglucosamine (UDP-GlcNAc) into UDP-N-acetylmannosamine (UDP-ManNAc). The latter is an intermediate in the biosynthesis of bacterial cell-surface polysaccharides and the enterobacterial common antigen (ECA). The ECA is a surface-associated glycolipid common to all members of the Enterobacteriacea family. In species such as Staphyloccocus aureus and Bacillus anthracis, the importance of the UDP-GlcNAc 2-epimerase in the biosynthesis of polysaccharides is highlighted by the presence of two functionally redundant copies of this enzyme. Studies support a mechanism in which the reaction proceeds through an initial anti-elimination of UDP to generate an intermediate 2-acetoamidoglucal, followed by the subsequent syn-addition of UDP to yield the product UDP-ManNAc.

This enzyme is allosterically regulated by its substrate UDP-GlcNAc. The structure of the ternary complex between the Bacillus anthracis UDP-GlcNAc 2-epimerase, its substrate UDP-GlcNAc and the reaction intermediate UDP, showed direct interactions between UDP and its substrate, and between the complex and highly conserved enzyme residues, identifying the allosteric site of the enzyme. The binding of UDP-GlcNAc is associated with conformational changes in the active site of the enzyme. Kinetic data and mutagenesis of the highly conserved UDP-GlcNAc-interacting residues confirm their importance in the substrate binding and catalysis of the enzyme. This constitutes the first example to our knowledge, of an enzymatic allosteric activation by direct interaction between the substrate and the allosteric activator.

In summary, the structure indicates that non-hydrolysing bacterial UDP-GlcNAc 2-epimerases use a new allosteric regulatory mechanism that involves direct interaction between one substrate molecule in the active site and another in the allosteric site. This regulatory mechanism helps the enzyme to hold its substrates and stable intermediates in the active site until the completion of a two-step elimination/addition reaction. The conservation of the allosteric site residues in the non-hydrolysing UDP-GlcNAc 2-epimerases indicates that this mechanism is used exclusively by this class of bacterial enzymes, thus providing a selective way of targeting them, particularly in the case of B. anthracis, for the development of novel antibiotic agents.

 

L.M. Velloso, S.S. Bhaskaran, R. Schuch, V.A. Fischetti, C.E. Stebbins.  (2008).  “A structural basis for the allosteric regulation of non-hydrolysing UDP-GlcNAc 2-epimerases.”  EMBO Rep 9(2):199-205. PMID: 18188181 [Abstract] [pdf] [pdb]

 

Drs. Lucas Velloso and Shyam Bhaskaran were the lead scientists on the epimerase project. Our collaborators were the group of Vincent Fischetti at the Rockefeller University.