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Introduction
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 |