JMol Tutorial created with support from Dr. Vincent Fischetti (Fischetti Lab, Rockefeller University) and Tim Herman (Center for Biomolecular Modeling).

Please email if you have any questions or to report problems with this tutorial.  Thank you.

Using this tutorial:
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The non-hydrolyzing bacterial UDP-GlcNAc 2-epimerase enzyme catalyzes the reversible conversion of UDP-N-acetylglucosamine (UDP-GlcNAc), into UDP N-acetylmannosamine (UDP-ManNAc) via an intermediate, 2-acetoamidoglycal. The reaction proceeds first with an anti-elimination of the UDP to create the intermediate, and then syn-addition yields the UDP-ManNac. UDP-ManNac is an intermediate in the biosynthesis of cell surface polysaccharides in bacteria as well as the enterobacterial common antigen, a surface-associated glycolipid.

UDP-GlcNAc binds in two locations in the enzyme, the active site and the allosteric site. X-ray crystallography consistently depicts the GlcNAc of the substrate in the allosteric site but not in the active site. The second UDP-GlcNAC allosterically regulates the enzyme and acts as an activator and induces conformational changes necessary to yield the final product.  In this structure, only the UDP of the substrate in the active site is displayed.

The non-hydrolyzing UDP-GlcNAc 2-epimerase is essential in bacterial cell wall development and highly conserved in strains including Staphylococcus aureus and Bacillus anthracis, both of which are pathogenic gram-positive strains. Study of this enzyme is crucial for future development of small molecule drugs that would allosterically inhibit binding of UDP-GlcNAc to inhibit bacterial growth.  The enzyme residues found to be coordinating the substrate, UDP-GlcNAc, are highly conserved in the non-hydrolyzing bacterial epimerase but not in their hydrolyzing mammalian counterparts.  This also makes this enzyme a favorable target in antibacterial drug development.

In the initial view of epimerase in this tutorial, the UDP in the active site and the UDP-GlcNAc in the allosteric regulatory site are displayed.  There are seven residues that play a role in binding the UDP-GlcNAc.  The five conserved allosteric site residues (Gln43, His44, His242, Arg210, and Gln70) have their backbones highlighted in green (residues conserved among non-hydrolyzing UDP-GlcNAc 2-epimerases).


Domains affected during conformational change
(script will only color domain, will not reset view)

When the allosteric regulator UDP-GlcNAc binds to epimerase, numerous important localized conformational changes occur in three loops: (His209-Gly215, Ile65-Leu72, and Val241-Pro245).  These changes are instrumental in converting the enzyme into an active state by positioning key residues around its substrate.

The loop formed by His209-Gly215 shifts the guadinium group of Arg210, allowing it to interact with the beta-phosphate of UDP (in the active site), a hydroxyl group on UDP-GlcNac (in allosteric site), the Glu136 sidechain, and water molecule coordinated by Glu212 and Glu136.  Thus Arg210 plays a crucial role in anchoring coordinating substrates both in the active site and the allosteric site. This loop also shifts His209, which makes hydrogen bonds to the beta-phosphate of the UDP in the active site.

The loop formed by Ile-65-Leu72 shifts and allows for the formation of 4 hydrogen bonds from Arg69 and Gln70 to the ribose and alpha-phosphate of UDP-GlcNac in the allosteric site.

The loop formed by Val241-Pro245 moves so His242 is in its bonding position to the beta-phosphate and a hydroxyl group on the UDP-GlcNac in the allosteric site.


View epimerase in spacefill

Clearly depicted in spacefill, the conformational changes from the binding of the UDP-GlcNAc in the allosteric site results in full enclosure of the UDP-GlcNAc in the active site (only the UDP seen here). This obstructs the active site’s access to the solvent, which prevents the products of anti-elimination reaction, UDP and 2-acetoamidoglucal, from diffusing away before they can react in syn-addition.


Highlight conserved residues and allosteric site

The allosteric site is surrounded by hydrophilic side chains. The UDP-GlcNAc in this extended pocket interacts with residues Gln43, Gln46, Gln70, His44, His242, Arg210, and Glu136.  Mutagenesis and kinetic data show the importance of these residues in the enzyme's binding and catalyzing abilities. UDP-GlcNAc in the allosteric site also forms hydrogen bonds with the alpha and beta phosphates of the UDP-GlcNAc (just the UDP seen in structure) in the active site to stabilize it through the second step of the epimerization reaction. Residues interacting with UDP-GlcNAc in the allosteric site are highly conserved in bacterial non-hydrolyzing epimerases but vary from hydrolyzing epimerases found in mammalian cells, making this a possible target for antimicrobial drug development.

Arg210 plays a significant role in the epimerization reaction. It stabilizes the both UDP-GlcNAc's in the active and allosteric sites. Arg210 also interacts with Glu136 through two hydrogen bonds. Arg210 is conserved in non-hydrolyzing bacterial UDP-GlcNAc 2-epimerases while Glu136 is conserved in all UDP-GlcNAc 2-epimerases, suggesting that the role of Arg210 and the conformational changes associated with it are unique to bacterial non-hydrolyzing UDP-GlcNAc 2-epimerases.

In addition, the His242 residue forms hydrogen bonds to the beta-phosphate and to a hydroxyl group of UDP-GlcNAc in the allosteric site.  His242 stabilizes the UDP intermediate in the active site, thus aids in the binding of UDP-GlcNAc to both active and allosteric sites. Gln70, Gln43 and His44 all form hydrogen bonds to the axial oxygens of the alpha-phosphate of UDP-GlcNAc in the allosteric site. His44 forms a hydrogen bond to an axial oxygen of the beta-phosphate of UDP-GlcNAc in the allosteric site, while Gln43 bonds to a carbonyl group of UDP-GlcNAc in the allosteric site.  Interaction between epimerase and the alpha phosphate of UDP-GlcNAc in the allosteric site help to position this substrate for optimal interaction with the UDP-GlcNAc in the catalytic site.


Highlight UDP in active site and UDP-GlcNAc in allosteric site

Epimerase binds a UDP-GlcNAc in both its active site and its allosteric ciste.  In this structure, only the UDP is seen in the active site.  The UDP-GlcNAc in the allosteric site is seen interacting with the seven key residues surrounding this pocket.

Hydrogen bonds between the two substrates help to keep them in place during the epimerization reaction. A ternary complex between UDP-GlcNAc 2-epimerase and the two substrates displays the first example of enzymatic allosteric activation through direct interaction between the substrate and allosteric activator.


View UDP and UDP-GlcNAc without enzyme


Model Designs

Wireframe model 1
protein backbone 300, protein sidechains in wireframe 250
substrates in wireframe 250
backbone of residues involved in conformational change colored blue
backbone of highly conserved residues colored green (overrides above blue)
residues interacting with allosteric substrate shown with sidechain

Wireframe model 2
same as above model but without substrates
substrates will be built separately


Tutorial and scripts prepared by Jonathan Ciriello, Yamini Nabar, and Neha Srivastava

PDB File: From V.Fischetti

Reference: Velloso LM, Bhaskaran SS, Schuch R, Fischetti VA, Stebbins CE. (2008) A structural basis for the allosteric regulation of non-hydrolysing UDP-GlcNAc 2-epimerases. EMBO Rep. Jan 11 (Epub).