Visualizing the reaction coordinate of an O-GlcNAc hydrolase

Article abstract of DOI:10.1021/ja9086769

(Chemical Presented) N-Acetylglucosamine β-O-linked to serine and threonine residues of nucleocytoplasmic proteins (O-GlcNAc) has been linked to neurodegeneration, cellular stress response, and transcriptional regulation. Removal of O-GlcNAc is catalyzed by O-GlcNAcase (OGA) using a substrate-assisted catalytic mechanism. Here we define the reaction coordinate using chemical approaches and directly observe both a Michaelis complex and the oxazoline intermediate. Copyright

Full text of DOI:10.1021/ja9086769

Published on Web 01/12/2010
Visualizing the Reaction Coordinate of an O-GlcNAc Hydrolase
Yuan He,† Matthew S. Macauley,‡ Keith A Stubbs,‡,§ David J Vocadlo,‡ and Gideon J Davies*,†
York Structural Biology laboratory, Department of Chemistry, The UniVersity of York, YO10 5YW, U.K.,
Department of Chemistry, Simon Fraser UniVersity, 8888 UniVersity DriVe, Burnaby, Canada, and Chemistry M313,
School of Biomedical, Biomolecular and Chemical Sciences, UniVersity of Western Australia, 35 Stirling Highway,
Crawley, Western Australia 6009, Australia
Received October 12, 2009; E-mail: davies@ysbl.york.ac.uk
O-GlcNAc1 is a dynamic post-translational modification critical
to life at the cellular level. Critical roles1 for O-GlcNAc in the
stress response, signal transduction, and transcriptional regulation2
including polycomb repression3,4 have been forwarded while
dysregulation of O-GlcNAc has been implicated in type II diabetes5
and neurodegeneration.6,7 The partial reciprocity of O-GlcNAc with
serine and threonine phosphorylation8 is notable in view of its
biological importance. Interestingly, in contrast to phosphorylation
where over 600 enzymes are involved in catalyzing its addition
and removal, only two enzymes regulate the O-GlcNAc modifica-
tion in metazoans: O-GlcNAc transferase (OGT) and O-GlcNAc
hydrolase (OGA).1 The dynamic nature of the O-GlcNAc modifica-
tion has made inhibitors of these two enzymes useful for altering
cellular levels of O-GlcNAc and interrogating the biological roles
of this modification. Small molecule chemical intervention has been
particular successful with OGA,9 a glycoside hydrolase family from
GH84 (one of >110 sequence based families of glycoside hydrolases
(www.cazy.org)). OGA catalyzes the removal of O-GlcNAc using
a substrate-assisted catalytic mechanism. Here we define the reaction
coordinate using chemical approaches and directly observe both
the Michaelis complex and the oxazoline intermediate.
The 3-D structures of two close bacterial homologues of human
OGA, NagJ10 from Clostridium perfringens and Bacteroides
thetaiotaomicrometer BtGH84,11 have had their 3-D structures
determined (reviewed in ref 12). Together with enzymatic data,11,13
these structures have solidified that both human and bacterial
enzymes use a catalytic mechanism in which the N-acetyl carbonyl
group is critical to catalysis, an indication of a two-step neighboring
group reaction mechanism that proceeds through a bicyclic oxazo-
line intermediate, Figure 1. The 3-D structures revealed an active-
center constellation of two adjacent carboxylates, which is a
signature motif of the growing set of glycoside hydrolases harness-
ing neighboring group participation.12 In the case of BtGH84, in
the first step of the reaction, “cyclization”, D243 plays the role of
general acid to aid departure of the leaving group while D242
activates the 2-acetamido group for nucleophilic attack; the net result
of these coordinated processes is formation of the oxazoline
intermediate, Figure 1. In the second step, “ring-opening”, D243
acts as a general base to aid attack of a water molecule at the
anomeric center while D242 aids opening of the oxazoline ring to
form the product with retained stereochemistry at the anomeric
center. To date, no analysis defining the complete set of stable
species along the reaction coordinate of any GH family known
to use substrate assisted catalysis (GH18, 20, 25, 56, 84, and
85)12 has been presented. In general, such structural elucidation
of species along the coordinates of enzyme catalyzed reactions
Figure 1. The catalytic mechanism of human OGA and family 84 glycoside
hydrolases proceeds via a two-step reaction mechanism though a Michaelis
complex shown here to have a distorted 1,4B/1S3 conformation and an
oxazoline intermediate having a 4C1 conformation. The catalytic residue
numbering shown is for BtGH84. For compound 1 (see Figure 2), R ) H
and X ) F; for 2, R ) F and X ) H; and for 3, R ) X ) H.
is a difficult but illuminating process, motivated by a desire to
enhance the basic understanding of enzyme catalysis and also
serving to stimulate the design of highly potent inhibitors (for
a recent perspective see ref 14).
A key species that defines the reaction coordinate of any enzyme-
catalyzed reaction is the Michaelis complex. Various approaches
to trap glycoside hydrolases with their unhydrolyzed substrates have
included nonhydrolyzable or slowly turned over substrates and/or
enzyme variants in which critical components of the catalytic
apparatus have been compromised.15 Recently a detailed linear free
energy analysis of human OGA demonstrated that when a substrate
contains a modest leaving group as well as a poor nucleophile, the
catalytic efficiency of the enzyme is synergistically impaired.16 We
reasoned that, under the right conditions, such a substrate might
be trapped in the active site unhydrolyzed. To this end, we tested
a wide range of such substrates and obtained co-crystals of BtGH84
and 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-ꢀ-D-glucopy-
ranoside16 1 (details of synthesis are in the Supporting Information,
SI online), Figure 2.
Figure 2. Compounds used in the visualization of the O-GlcNAcase
reaction coordinate. (1) 3,4-Difluorophenyl 2-deoxy-2-difluoroacetamido-
ꢀ-D-glucopyranoside, (2) 2-acetamido-2-deoxy-5-fluoro-ꢀ-D-glucopyranosyl
fluoride, and (3) 4-methylumbelliferyl 2-acetamido-2-deoxy-ꢀ-D-glucopy-
ranoside.
We solved the 3-D structure at 2.2 Å resolution (methods in SI,
refinement statistic in Supplemental Table 1) harnessing the 2-fold
noncrystallographic symmetry to reveal an unambiguous density
for unhydrolyzed 1 within the active site, Figure 3a. Strikingly,
† The University of York.
‡ Simon Fraser University.
§ University of Western Australia.
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C O M M U N I C A T I O N S
analogy with other glycosidase systems, it was reasoned that
incorporation of a very good fluoride leaving group would make
the intermediate kinetically accessible and make the breakdown
of the oxazoline intermediate relatively slow.15,19 Despite these
considerations, turnover of the putative 5-fluoro-2′-methyl-R-D-
glucopyrano-[2,1-d]-∆2′-oxazoline (5F-oxazoline) intermediate by
the wild-type enzyme was likely too rapid and we were unable to
observe this species by X-ray crystallography. Instead, we used 2
in conjunction with a variant of BtGH84 in which the general acid/
base is impaired (D243N) (Figure 1). We anticipated that the
absence of the general acid/base would leave the cyclization step
unaffected since departure of the fluoride ion leaving group cannot
benefit from general acid catalysis. The ring-opening of the
intermediate, however, would be slowed since the attack of water
on the anomeric center is promoted by general base catalysis. This
strategy succeeded and enabled accumulation and observation of
the oxazoline in soaked and flash-frozen crystals, Figure 3b. We
4
find the 5F-oxazoline intermediate binds in an approximate C1
conformation.
The second strategy enabled us to observe a chemically
“unmodified” oxazoline intermediate. A comparison of the set of
GH families using substrate assisted catalysis reveals two key
residues: a general acid/base (here D243) and a second group which
also likely serves as a general acid/base to aid formation and
breakdown of oxazoline intermediate (here D242), Figure 1. In all
families except GH85, this second residue, that interacts directly
with the oxazoline intermediate, is an Asp or Glu; however, it was
recently shown that in GH85 an Asn can also support catalysis.
Notably, these GH85 enzymes are efficient catalysts for biosyn-
thesis, and this may be due to the oxazoline intermediate being
more stable and favoring partitioning to acceptor sugars more
efficiently than to water. With this in mind, we speculated that the
analogous D242N variant of BtGH84 might retain activity but that
the ring-opening step of the reaction may be slowed preferentially.
Figure 3. Visualization of the reaction coordinate of O-GlcNAc hydrolases.
(a) Michaelis complex of 1 (shown with gray bonds) bound within the wild-
type BtGH84 glycoside hydrolase active site. (b) The 5F-oxazoline
intermediate obtained from treating D243N BtGH84 with 2. The oxazoline
intermediate obtained from treating D242N BtGH84 with 3. Electron
densities are maximum likelihood weighted 2Fobs - Fcalc synthesis contoured
(approximately 0.4 e-/Å3) in divergent stereo.
Specifically, we considered that departure of a fairly good phenol
leaving group might still be fairly efficient but that breakdown of
the oxazoline might be slower. Steady-state kinetics (Supporting
Information) using a substrate with a 4-methylumbelliferone leaving
group, namely 3, showed that the D242N mutant was active on
this substrate, albeit with an efficiency ∼320-fold times lower than
that of wild-type enzyme. At a pH value of 8.5 we find a 12-fold
reduction in apparent Km (1 mM to 0.08 mM, SI Figure 1),
suggesting that the second step of the reaction may be the rate-
determining step for this mutant enzyme (since Km ) [E][S]/∑[E-
bound species], a low Km suggests accumulation of an enzyme
bound intermediate). In this scenario, departure of the activated
leaving group enables cyclization to form the oxazoline intermedi-
ate, but breakdown of the intermediate by ring-opening is slow since
protonation of the oxazoline either on enzyme by D242N or in
solution is likely not thermodynamically favorable. This hypothesis
predicts that presteady state stopped flow fluorescence kinetics
should reveal a burst phase arising from accumulation of the
oxazoline intermediate followed by a steady state turnover of this
species. We find precisely such kinetic behavior occurring when
the enzyme is initially mixed with the substrate, Figure 4a. Further
consistent with this hypothesis is that the magnitude of the burst
matched the enzyme concentration (burst size is 94% of [E]o).
Accordingly, when we soaked crystals of the D242N variant grown
at pH 8.5 with 3, we observe an intact trapped oxazoline
intermediate. Like the 5F-oxazoline, the unsubstituted oxazoline
binds in a 4C1 chair conformation, Figure 3c. Indeed, beyond a slight
shift in the position of Lys166 away from the O3 atom of the
D242N oxazoline complex that likely reflects the elevated pH used
the pyranose ring of the substrate adopts a conformation between
1,4B and 1S3, Figure 1, differing significantly from the 4C1
conformation preferred in solution but consistent with trapped
Michaelis complexes on GH18 chitinases17 and GH20 chitobiases18
that also share the neighboring group mechanism.12 The distorted
conformation we observe here, however, is consistent with work
conducted on other glycosidases that act on a ꢀ-glycosidic linkage
via a retaining catalytic mechanism involving formation of a
covalent glycosyl enzyme intermediate.15 Such a conformation
places the leaving group C1-OR bond in a pseudoaxial position
“above” the plane of the pyranose ring. As a result, the N-acetyl
carbonyl oxygen of the substrate itself lies poised to displace the
leaving group from the tetrahedral anomeric center, with an
Onuc-C1 distance of 3.4 Å and an Onuc-C1-Olg angle of 170°
(Supplemental Movie 1).
Direct observation of enzyme bound intermediates has also
proven difficult. For glycoside hydrolases, different approaches have
been used to trap covalent glycosyl enzyme intermediates.19
A
bicylic oxazoline intermediate has never been observed, however,
for any glycoside hydrolase using substrate-assisted catalysis,
perhaps because these species are extremely reactive or perhaps
because they are noncovalently bound to these enzymes. To
surmount this problem and visualize the putative bicylic oxazoline
intermediate we devised two different strategies. In the first strategy
we used 2-acetamido-2-deoxy-5-fluoro-ꢀ-D-glucopyranosyl fluo-
ride20 2, Figure 2, since we speculated that the electronegative
fluorine substituent adjacent to the endocyclic oxygen (O5) would
slow both the formation and breakdown of the oxazoline intermedi-
ate by destabilizing the flanking cationic transition states. By
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Such observations are consistent with Whitworth’s proposal made for
human OGA that a broad high energy series of species encompassing
a late transition state are found along the reaction coordinate.23 The
description of a 1S3T4H3T4C1 pathway for oxazoline formation,
presented here, coupled to solution studies on OGA transition-state
poise23 therefore provides the first sighting of an oxazoline intermediate
bound to any glycoside hydrolase using substrate assisted catalysis
and coherently defines the conformational itinerary of this class of
enzymes.
Acknowledgment. This work was funded by the Biotechnology
and Biological Sciences Research Council (BBSRC) and the
Canadian Institutes of Health Research and the Natural Sciences
and Engineering Research Council of Canada (NSERC). G.J.D. is
a Royal Society-Wolfson Research Merit award recipient. D.J.V.
is a Canada Research Chair in chemical glycobiology and a Scholar
of the Michael Smith Foundation for Health Research (MSFHR).
M.S.M. holds scholarships from NSERC and the MSFHR.
Supporting Information Available: Details of organic synthesis
of compound 1, structure solution and refinement and steady and
presteady-state kinetics. Movie of the Michaelis complex of BtGH84
with 1 and schematic diagram of interactions of the compound 3 derived
oxazoline intermediate. This material is available free of charge via
the Internet at http://pubs.acs.org.
Figure 4. Electrophilic migration21 along the reaction coordinate of
O-GlcNAc hydrolases. (a) Presteady state kinetics for the hydrolysis of 3
with the D242N variant at pH 8.5. (b) Overlap of the D242N trapped
oxazoline intermediate (gray) overlaid with the Michaelis complex of
unhydrolysed 1. (c) Simplified schematic representation of the “electrophilic
migration” of the anomeric carbon from the Michaelis complex (1,4B/1S3
conformation) through to the oxazoline intermediate in the 4C1 conformation.
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with Asp242 and Asn339 respectively (Supplemental Figure 2). In
both trapped intermediates a potential hydrolytic water (colored
purple in Figure 3b,c) lies ∼3.6 Å above the anomeric carbon poised
for nucleophilic attack.
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