Structure of a class I tagatose-1,6-bisphosphate aldolase: Investigation into an apparent loss of stereospecificity

Article abstract of DOI:10.1074/jbc.M109.080358

Tagatose-1,6-bisphosphate aldolase from Streptococcus pyogenes is a class I aldolase that exhibits a remarkable lack of chiral discrimination with respect to the configuration of hydroxyl groups at both C3 and C4 positions. The enzyme catalyzes the reversible cleavage of four diastereoisomers (fructose 1,6-bisphosphate (FBP), psicose 1,6-bisphosphate, sorbose 1,6-bisphosphate, and tagatose 1,6-bisphosphate) to dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate with high catalytic efficiency. To investigate its enzymatic mechanism, high resolution crystal structures were determined of both native enzyme and native enzyme in complex with dihydroxyacetone-P. The electron density map revealed a (α/β)₈ fold in each dimeric subunit. Flash-cooled crystals of native enzyme soaked with dihydroxyacetone phosphate trapped a covalent intermediate with carbanionic character at Lys ²⁰⁵, different from the enamine mesomer bound in stereospecific class I FBP aldolase. Structural analysis indicates extensive active site conservation with respect to class I FBP aldolases, including conserved conformational responses to DHAP binding and conserved stereospecific proton transfer at the DHAP C3 carbon mediated by a proximal water molecule. Exchange reactions with tritiated water and tritium-labeled DHAP at C3 hydrogen were carried out in both solution and crystalline state to assess stereochemical control at C3. The kinetic studies show labeling at both pro-R and pro-S C3 positions of DHAP yet detritiation only at the C3 pro-S-labeled position. Detritiation of the C3 pro-R label was not detected and is consistent with preferential cis-trans isomerism about the C2-C3 bond in the carbanion as the mechanism responsible for C3 epimerization in tagatose-1,6-bisphosphate aldolase.

Full text of DOI:10.1074/jbc.M109.080358

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 27, pp. 21143–21152, July 2, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Structure of a Class I Tagatose-1,6-bisphosphate Aldolase
□
S
INVESTIGATION INTO AN APPARENT LOSS OF STEREOSPECIFICITY*
Received for publication, November 6, 2009, and in revised form, February 28, 2010 Published, JBC Papers in Press, April 28, 2010, DOI 10.1074/jbc.M109.080358
Clotilde LowKam, Brigitte Liotard, and Jurgen Sygusch1
From the Department of Biochemistry, Universite´ de Montre´al, Montre´al, Que´bec H3C 3J7, Canada
(2–4). Tagatose-1,6-bisphosphate (TBP)2 aldolase is an induc-
ible enzyme that, although demonstrating greatest affinity for
D-tagatose 1,6-bisphosphate, can also use as substrate the
bisphosphorylated D-hexose stereoisomers: sorbose bisphos-
phate, psicose bisphosphate, and fructose bisphosphate (5).
The four sugars are diastereoisomers and differ in stereochem-
istry at carbon 3 and at carbon 4 with respect to the configura-
tion of their hydroxyl groups. The cleavage of the four sugars
produces glyceraldehyde 3-phosphate and dihydroxyacetone
phosphate (DHAP), whereas the condensation of glyceralde-
hyde 3-phosphate and DHAP produces a mixture of the four
D-hexoses in Staphylococcus aureus (5).
Tagatose-1,6-bisphosphate aldolase from Streptococcus pyo-
genes is a class I aldolase that exhibits a remarkable lack of chiral
discrimination with respect to the configuration of hydroxyl
groups at both C3 and C4 positions. The enzyme catalyzes the
reversible cleavage of four diastereoisomers (fructose 1,6-
bisphosphate (FBP), psicose 1,6-bisphosphate, sorbose 1,6-
bisphosphate, and tagatose 1,6-bisphosphate) to dihydroxyac-
etone phosphate (DHAP) and D-glyceraldehyde 3-phosphate
with high catalytic efficiency. To investigate its enzymatic
mechanism, high resolution crystal structures were determined
of both native enzyme and native enzyme in complex with dihy-
droxyacetone-P. The electron density map revealed a (␣/␤)8
fold in each dimeric subunit. Flash-cooled crystals of native
enzyme soaked with dihydroxyacetone phosphate trapped a
covalent intermediate with carbanionic character at Lys205, dif-
ferent from the enamine mesomer bound in stereospecific class
I FBP aldolase. Structural analysis indicates extensive active site
conservation with respect to class I FBP aldolases, including
conserved conformational responses to DHAP binding and con-
served stereospecific proton transfer at the DHAP C3 carbon
mediated by a proximal water molecule. Exchange reactions
with tritiated water and tritium-labeled DHAP at C3 hydrogen
were carried out in both solution and crystalline state to assess
stereochemical control at C3. The kinetic studies show labeling
at both pro-R and pro-S C3 positions of DHAP yet detritiation
only at the C3 pro-S-labeled position. Detritiation of the C3
pro-R label was not detected and is consistent with preferential
cis-trans isomerism about the C2–C3 bond in the carbanion as
the mechanism responsible for C3 epimerization in tagatose-
1,6-bisphosphate aldolase.
Aldolases are broadly categorized with respect to their catalytic
mechanism into two classes. Class I aldolases are characterized by
formation of covalent Schiff base intermediates (6–8), whereas
class II aldolases are metallodependent enzymes and use a divalent
transition metal ion to polarize the substrate ketose (9, 10). Of all
aldolases, class I FBP aldolase from rabbit muscle has been the
most extensively studied (11, 12). To form the FBP C3–C4 bond in
aldol condensation, the enzyme stereospecifically abstracts the
pro-S C3 proton of the iminium intermediate formed by a lysine
residue in the active site with DHAP (13–15), thereby generating
the carbanionic character at C3 of DHAP for the aldol reaction.
The nascent carbon-carbon bond has the same orientation as the
pro-S␣-hydrogen initially abstracted from the DHAP imine inter-
mediate, the carbanion si face being subjected to a nucleophilic
attack by the si face of the incoming glyceraldehyde 3-phosphate
aldehyde (16). The iminium intermediate formed is then hydro-
lyzed, andFBPisreleased. Similarly, in L-rhamnulose-1-phosphate
aldolase, the attack by the re face of L-lactaldehyde on the re face of
DHAP yields rhamulose 1-phosphate (17, 18) and also retains
configuration.
Racemic aldol condensation products with respect to the
configuration at C4 have been noted in a number of aldolases.
In 2-keto-4-hydroxyglutarate aldolase (19) and in L-fuculose-1-
phosphate aldolase, racemization occurs at C4 of the conden-
sation products, resulting from random orientation of the alde-
hyde during stereofacial attack (20) and from its chemical
nature (21). In contrast, configuration at both C3 and C4 is
not retained in the catalytic mechanism of the class I TBP
aldolase from S. aureus, given that aldol condensation yields
a mixture of sorbose bisphosphate, psicose bisphosphate, fruc-
Aldolases are crucial enzymes in living organisms because of
their role in essential metabolic pathways, such as gluconeo-
genesis and glycolysis. Their ability to control the stereochem-
istry of the carbon-carbon bond formation makes them models
for de novo preparation of carbohydrates (1) and ideal alterna-
tives to traditional methods in synthetic organic chemistry
* This work was supported by grants from the Natural Science and Engineer-
ing Research Council (Canada) and Canadian Institutes for Health Research
(to J. S.).
The on-line version of this article (available at http://www.jbc.org) contains tose bisphosphate, and tagatose bisphosphate (5).3 Attempts
□S
supplemental Fig. S1 and Table S1.
Theatomiccoordinatesandstructurefactors(codes3MHFand3MHG)havebeen
deposited in the Protein Data Bank, Research Collaboratory for Structural 2 The abbreviations used are: TBP, tagatose 1,6-bis(phosphate); FBP, fructose
Bioinformatics, Rutgers University, NewBrunswick, NJ(http://www.rcsb.org/).
1,6-bis(phosphate); DHAP, dihydroxyacetone phosphate; MAD, multiple-
wavelength anomalous dispersion; r.m.s., root mean square.
1 To whom correspondence should be addressed: CP 6128, Station Centre
Ville, Montre´al, Quebec H3C 3J7, Canada. Tel.: 514-343-2389; Fax: 514-343- 3 This observation was verified for S. pyogenes TBP aldolase and is described
6463; E-mail: Jurgen.Sygusch@UMontreal.ca.
in the supplemental material.
JULY 2, 2010•VOLUME 285•NUMBER 27
JOURNAL OF BIOLOGICAL CHEMISTRY 21143

Structure of a Class I TBP Aldolase
have been made to switch the specificity of class II TBP aldola- aldolase was eluted using a continuous gradient of NaH2PO4.
ses; however, these have been met with limited success (22, 23). The active fractions were then dialyzed overnight against a pre-
Three-dimensional structures of class I FBP aldolases have cipitating solution of 90% saturating (NH4)2 SO4, pH 7.0, and
been determined from different organisms (24–26), and reac- then applied after resolubilization onto a size exclusion column
tion intermediates have been characterized (27), providing a (SuperdexTM 200, Amersham Biosciences). The purified pro-
detailed description of the catalytic mechanism at the molecu- tein was precipitated overnight using the same 90% saturated
lar level, including a structural explanation for stereospecific (NH4)2 SO4, pH 7.0 solution as before, and stored at 4 °C. Yields
proton exchange at C3 (11). In sharp contrast, insights into how corresponded to ϳ50 mg of purified enzyme per liter of E. coli
class I TBP aldolase catalyze racemic aldol condensation prod- culture.
ucts at the molecular level have been hampered by an absence of
TBP aldolase crystals were grown using the hanging drop
structures of reaction intermediates. We thus initiated high res- method, from a 1:1 mixture of protein solution (initial concen-
olution crystallographic studies of class I TBP aldolase from tration 5 mg/ml in 10 mM Tris-HCl, pH 7.0) and precipitant
Streptococcus pyogenes in conjunction with isotopic labeling buffer (9–11% polyethylene glycol 4000 in 0.2 M calcium acetate
studies to investigate its reaction mechanism and the structural and 0.1 M Tris-acetic acid, pH 7.5) that was equilibrated against
basis for the apparent loss of stereospecificity.
a reservoir of precipitant.
Here, we present the three-dimensional structure of a native
Cleavage Assay—The substrate cleavage rate was determined
class I TBP aldolase as well as its complex with DHAP using by measuring the decrease in A340/min using a coupled assay
TBP aldolase crystals incubated in the presence of DHAP. Flash (27). Aldolase was diluted in 50 mM Tris-HCl, pH 7.5, and
cooling of a native TBP aldolase crystal soaked in a saturating added to a cuvette containing 50 mM Tris-HCl, pH 7.5, 10 mM
DHAP solution trapped a covalent reaction intermediate. The EDTA, 0.16 mM NADH, and 10 ␮g/ml glycerol-3-phosphate
structure revealed conformational changes upon binding, dehydrogenase/triose-phosphate isomerase. Assays (1 ml) were
whereby the active site undergoes asymmetrical narrowing to performed in triplicate at 23 °C following the addition of sub-
grasp DHAP. The location of an invariant water molecule strate. The cleavage rate for FBP and TBP substrates was mea-
within close contact of the DHAP C3 carbon in the trapped sured over a substrate concentration range of 1–5000 ␮M, using
intermediate underscored a stereospecific proton transfer 1 ␮g of wild type enzyme. TBP aldolase concentration was
mechanism conserved in Schiff base-forming aldolases.
determined by BCA protein assay reagent (Pierce), with bovine
The extent of stereochemical control in class I TBP aldolases serum albumin serving as a standard.
with respect to C3 epimerization was also examined in both
Data Collection and Processing—TBP aldolase crystals were
solution and crystalline state. Catalytic proton transfer was soaked in DHAP buffer (mother liquor plus 5 mM DHAP) for 5
assessed by the lability of the pro-R and pro-S C3 hydrogens of min. Crystals were then mounted in nylon cryoloops (Hampton
DHAP to exchange in tritiated water. The proton exchange Research) after brief immersion in cryoprotectant (mother liq-
studies, in conjunction with the structural data, afforded iden- uor plus 5 mM DHAP plus 20% glycerol) and then immediately
tification of the carbanion as the dominant steady state reaction flash-cooled in a nitrogen gas stream at 100 K. Diffraction data
intermediate and indicated cis-trans isomerism about the car- were collected at beamline X25 of the National Synchrotron
banion C2–C3 bond in explaining the loss of stereospecific pro- Light Source (Brookhaven National Laboratory, Upton, NY). A
ton exchange at the DHAP C3 carbon in TBP aldolase. These native data set was also collected at beamline X29. Data index-
findings stand in contrast to the highly stereospecific proton ing and integration were performed using HKL2000 (30).
exchange mechanism found in class I FBP aldolases and point
MAD Phasing and Refinement—The three-dimensional
to rotational isomerization in specific reaction intermediates as structure of TBP aldolase from S. pyogenes was solved using
an evolutionary simple solution capable of generating enantio- MAD phasing on previously collected data of a SeMet crystal
meric promiscuity.
(28). The three different wavelength (inflection, peak, and
remote) MAD data sets allowed the program SOLVE (31) to
determine the positions of five selenium atoms of six possible in
EXPERIMENTAL PROCEDURES
Purification and Crystallization—Plasmid pKK-223-3 with each protomer and to estimate initial protein phases. Solvent
T7 isopropyl 1-thio-␤-D-galactopyranoside-inducible pro- flattening with the program RESOLVE (32) yielded an inter-
moter and coding for the lacD2 gene product from S. pyogenes, pretable electron density map calculated at 2.6 Å resolution.
tagatose-1–6-bisphosphate aldolase 2, was transformed and Iterative cycles of chain tracing using O (33) and model refine-
overexpressed using the JM109 strain (Promega) in Escherichia ment using the Crystallography and NMR System (CNS) (34)
coli. Recombinant TBP aldolase was purified to homogeneity defined a trace of the polypeptide backbone that refined to
using a three-step chromatographic protocol as reported pre-
viously (28). Briefly, cells were lysed at 4 °C, and the lysate was
R
cryst (Rfree) values of 0.246 (0.290).
Molecular Replacement and Refinement—Native free TBP
applied onto an anion exchange column (DEAE-Sepharose Fast aldolase crystallized in the P212121 space group with four pro-
Flow) and eluted with a NaCl gradient at pH 7.5. Enzymatic tomers in the asymmetric unit and diffracted to 1.87 Å resolu-
activity was determined by following NADH oxidation at 340 tion. The structure was solved by molecular replacement using
nm using a coupled assay (29). The active fractions were dia- the initial MAD phased model as a search probe. Structure
lyzed overnight against a solution of 1.5 M (NH4)2 SO4, 50 mM refinement consisted of iterative rounds of simulated annealing
NaH2PO4, pH 7.0, and then applied onto a hydrophobic affinity and minimization with CNS and Phenix (35) and model build-
column (phenyl-Sepharose, Amersham Biosciences). TBP ing using O and Coot (36). Water molecules were initially
21144 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 27•JULY 2, 2010

Structure of a Class I TBP Aldolase
TABLE 1
S.D. values and were estimated based on their value in each
aldolase protomer unless specified otherwise. Intersubunit var-
iability among the four protomers in the asymmetric unit cell
was analyzed by the program Polypose (40) and yielded r.m.s.
differences, based on C␣ atom coordinates, similar to the error
in the atomic coordinates for each structure. All figures were
prepared using the program PyMOL (41).
Data collection and refinement statistics
Native
DHAP-bound
Data collection
Resolution
50-1.87 (1.97-1.87)a 50-1.92 (2.02-1.92)
Wavelength
0.9795
144,212 (15,756)
74.8 (82.5)
0.9795
142,361 (12,153)
85.9 (74.1)
Unique reflections
Completeness (%)
Average I/␴(I)
7.1 (1.4)
7.8 (2.9)
b
c
Rsym
0.092 (0.513)
0.071 (0.419)
0.113 (0.650)
0.101 (0.326)
0.045 (0.186)
0.120 (0.440)
Chemical identity of the enzymatic intermediate trapped in
the DHAP-TBP aldolase structure was determined with the
program O (33), using a simulated annealing omit map. The
real space statistic Rfact was calculated in O (RS_FIT function)
to evaluate the fit of Lys205 N␨ and C⑀ atoms, and DHAP C1, C2,
C3, and O3 atoms were modeled as enamine and iminium
forms to the election density. The discrimination between
those two chemical species was made using Student’s t test
comparing pairwise Rfact statistics in each subunit, with bound
DHAP refined as either iminium/carbanion or enamine form
(11).
Rpim
d
Rmeas
Unit cell parameters
a, b, c (Å)
63.7, 106.2, 238.5
64.1, 108.2, 238.7
Refinement
No. of atoms
Protein
10,248
2336
7 Ca
1
10,192
Water
2597
Hetero
7 Ca, 36 DHAP atoms
␴ cut-off; I/␴(I) Ͼ
1
16.1
19.9
R
R
cryst (%)e
free (%)f
17.5
21.8
r.m.s. deviation
Bond length (Å)
Bond angle (degrees)
Average B-factor (Å )
Ramachandran analysisg (%)
Favored
0.005
0.929
32.75
0.004
0.869
22.22
Proton Exchange—Tritium was used as a tracer to measure
label incorporation at the DHAP C3 hydrogen catalyzed by TBP
aldolase in solution and in crystals. Incorporation of the label at
(S)-C3 and (R)-C3 of DHAP was determined using an ion
96.3
3.7
97.1
2.9
Allowed
a Values in parentheses are given for the highest resolution shell.
b
Ϫ
R
sym ϭ ͚ ͚ ͉Ii(hkl) Ϫ Ii(hkl)͉/͚ ͚iIi(hkl) with i running over the number of
hkl i hkl
exchange protocol (Dowex Cl resin) described previously
independent observations of reflection hkl.
c Rpim ϭ ͚ ͌(1/(n Ϫ 1)) ͚i (͉Ii Ϫ Imean͉)/͚ ͚i(Ii).
(16). Stereospecific deprotonation at DHAP C3 by rabbit mus-
cle aldolase and triose-phosphate isomerase was used to deter-
mine the extent of stereospecific labeling as (S)-[3-3H1]DHAP
and (R)-[3-3H1]DHAP, respectively (16). Label released as triti-
ated water (detritiation) was determined as described previ-
ously and quantitated to assess epimerization at C3 by TBP
aldolase.
hkl
hkl
hkl
d Rmeas ϭ ͚ ͌(n/(n Ϫ 1)) ͚i (͉Ii Ϫ Imean͉)/͚ ͚i(Ii).
e Rcryst ϭ ͚hklʈFo(hkl)͉ Ϫ ͉Fc(hkl)ʈ/͚ ͉Fo(hkl)͉.hkl
hkl
hklÎT
f Rfree ϭ ͚hklÎTʈFo(hkl) Ϫ ͉Fc(hkl)ʈ/͚
͉Fo(hkl)͉, where T is a test data set randomly
selected from the observed reflections prior to refinement. The test data set was
not used throughout refinement and contains 7.5% of the total unique reflections
for native and native aldolase bound with DHAP.
g Analyzed by MolProbity (34).
added automatically by CNS and Phenix and by manual addi-
DHAP labeled as (S)-[3-3H1]DHAP or (R)-[3-3H1]DHAP
tion in subsequent rounds. The final model has in each asym- from tritiated water (tritiation) was prepared using the ability of
metric unit cell four protomers of 322 residues each, 2336 water FBP aldolase and triose-phosphate isomerase to stereospecifi-
molecules, and 7 cations of calcium.
cally label pro-S and pro-R C3 positions of DHAP, respectively.
The liganded crystal structure was isomorphous with the Detritiation catalyzed by TBP aldolase in solution and in crys-
crystal structure of the native aldolase and diffracted to 1.92 Å tals was followed by the appearance of label as tritiated water
resolution. Initial phases used for model building of the ligan- from (S)-[3-3H1]DHAP or (R)-[3-3H1]DHAP using the same
ded structure were obtained by molecular replacement using ion exchange protocol. Rates of label exchange were deter-
the MAD phased model for the TBP aldolase as a search probe. mined under conditions corresponding to initial rate kinetics.
The polypeptide trace of the liganded structure was determined
Aliquots of soluble TBP aldolase ranging from 1 to 5 ␮g made
using the same model building and refinement protocol as up in 10 mM Tris-HCl, 100 mM NaCl, pH 7.0, were used for the
described for the native enzyme. DHAP was refined as a cova- exchange studies. For the tritiation experiments, 20 mM DHAP
lent intermediate bound to Lys205 in all four protomers.
and 5 mCi of T2O (average specific activity of 70884 cpm/␮mol)
Structure refinement used all reflections having an I/␴(I) Ͼ 1; were added to the mixture and incubated for 1–10 min at room
however, electron density maps were calculated to the resolu- temperature. For the detritiation experiments, 4 mM DHAP
tion indicated in Table 1 to ensure at least ϳ80% completeness and 1 mM of DHAP labeled at either the pro-R or pro-S position
in the highest resolution shell with an I/␴(I) Ͼ 2. The PRODRG of carbon C3 (average specific activity of 2053 cpm/␮mol), were
server was used to generate ligand topology and parameter files added and incubated for 1–10 min at room temperature. For
(37). The presence of ligands in the final models was confirmed tritiation and detritiation by TBP aldolase in the crystalline
by inspection of simulated annealing Fo Ϫ Fc omit maps. Final state, crystals were washed three times in fresh mother precip-
model statistics, calculated with CNS and MolProbity are itant solution and then incubated in the same tritiation and
shown in Table 1. Multiplicity-weighted R factors (38) as out- detritiation mixtures as was the soluble protein. Quantities of
put by SCALA in CCP4 (39) are also shown in Table 1. The final crystalline TBP aldolase used ranged from 38 to 51 ␮g, and
structure models have Rcryst (Rfree) values of 0.175 (0.218) and incubation times ranged from 10 min to 4 h. The quantity of
0.161 (0.199), respectively. The corresponding positional errors crystalline enzyme and incubation time was greater compared
in atomic coordinates using maximum likelihood plots were with soluble TBP aldolase to ensure sufficient count rates that
estimated to be 0.21 and 0.17 Å, respectively. Errors in hydro- were significantly above background. Net count rates were cal-
gen bond distances and positional differences are reported as culated by subtracting background count rate measured in the
JULY 2, 2010•VOLUME 285•NUMBER 27
JOURNAL OF BIOLOGICAL CHEMISTRY 21145

Structure of a Class I TBP Aldolase
absence of enzyme. Background represented Ͻ0.1% of the low-
est exchange rate measured in the presence of soluble enzyme
and between 25 and 50% of the count rate in the presence of
crystalline enzyme. The final essay volume was 1 ml, and 300-␮l
aliquots were used for analysis. Exchange rates were measured
in triplicate with both soluble and crystalline enzyme.
Kinetic Solvent Isotope Effect—To evaluate D2O kinetic sol-
vent isotope effects, enzyme activity measurements were per-
formed in D2O using 50 mM Tris-acetic acid buffer, pH 7.5. The
effect of D2O on pH was corrected for. Substrate and Tris-
acetic acid buffer was made up in D2O, and coupling enzymes
were diluted in D2O prior to usage. Together, this amounted to
less than a 5% reduction in D2O concentration in the activity
assay. TBP aldolase was dialyzed against three successive
changes of D2O for at least 1 h until measured activity became
stable. Enzyme dialyzed against three similar successive
changes of H2O was used for control.
FIGURE 1. A, covalent enzymatic intermediate formed by S. pyogenes TBP
aldolase with DHAP. DHAP P1 oxyanion makes interactions with Arg278
,
Gly277, and Ser249, and DHAP C3 via its hydroxyl interacts with Asp27 and
Lys125. WatermoleculeWat1 (W1)makesclosecontactwithDHAPC3hydroxyl
and hydrogen-bonds with Glu163, whereas Wat2 (W2) interacts with the DHAP
oxyanion and makes close contacts with Wat1, Glu163, and Arg278. Difference
electron density was calculated from a 1.92 Å simulated annealing Fo Ϫ Fc
omit map encompassing Lys205 and DHAP and contoured at 0.0 ␴. B, electron
density showing trapping of a carbanion intermediate in the active site of TBP
aldolase. Electron density of the enzymatic intermediate was consistent with
geometrycorrespondingtoacarbanionand/oriminiumspeciesboundinthe
active site of all subunits of the trapped DHAP-TBP aldolase complex. The
covalent intermediate has sp2 hybridization at Lys205 N␰ and hence planar
shape when viewed perpendicular to the plane containing DHAP carbon
atoms C1, C2, and C3; additionally, DHAP O3 points out of this plane, corrob-
orating the intermediate identification as a carbanion and/or iminium. Differ-
ence electron density was calculated from a 1.92 Å simulated annealing Fo Ϫ
Fc omit map encompassing Lys205 and DHAP and contoured at 3.0 ␴. Green
dashes, hydrogen bonding interactions; red spheres, water molecules.
RESULTS
Native TBP Aldolase—TBP aldolase from S. pyogenes exhib-
its a ␣/␤8-triose-phosphate isomerase barrel fold similar to the
polypeptide fold observed for rabbit muscle aldolase (24), with
which it shares less than 12% sequence identity. The secondary
structure elements assigned using PyMOL consist of 11 ␣-heli-
ces and eight ␤-strands. The native enzyme has four protomers
in the asymmetric unit cell, and arrangement of the protomers
is best described in terms of two identical dimers. Each dimer
interface implicates primarily hydrogen bond interactions
between subunit ␣ helices, ␣5 and ␣6 of one subunit and the
same helices ␣5 and ␣6 in the other subunit. The surface buried
at the subunit dimer interface represents 1908 Å2, whereas the
dimer-dimer interface, constituted by one dimer subunit inter- aldolase subunit (Fig. 1B). Average B-factors of the bound
acting with the subunit dimer interface of the second dimer, DHAP (16.6 Ϯ 6.2 Å2) and of interacting side chains (14.3 Ϯ 5.8
buries 1319 Å2. Calcium ions were located at the dimer-dimer Å2) indicate full active site occupancy by DHAP.
interfaces and participated in bridging interactions. Size exclu-
To further corroborate the identity of the bound intermedi-
sion chromatography corroborated the dimeric quaternary ate, the real space Rfact statistic was calculated using the O pro-
structure of TBP aldolase, which elutes at a relative molecular gram in order to gauge the model fit to the electron density. The
weight of 70,000. A schematic representation of the TBP aldol- derived Student’s t test compared Rfact values for DHAP mod-
ase dimer with overall dimensions of ϳ34 ϫ 36 ϫ 44 Å is shown eled either as an enamine intermediate with planar geometry
in supplemental Fig. S1. The dimeric subunits are related by a for DHAP atoms C1, C2, C3, and O3 and sp3 hybridization at
non-crystallographic 2-fold axis of rotation such that subunit Lys205 N␨ or as an iminium/carbanion intermediate with sp2
active sites face the same direction.
hydridization at Lys205 N␨ and coplanarity for DHAP atoms C1,
Carbanion Intermediate—A TBP aldolase crystal soaked in a C2, and C3 only. The statistic was discriminatory with p ϭ
DHAP solution and flash-cooled trapped a covalent enzymatic 0.072, the iminium and/or carbanion showing significantly
intermediate in each of the four subunits in the asymmetric unit lower Rfact than the enamine in all four subunits.
that permitted unambiguous identification of the active site.
The DHAP intermediate is engaged in several stabilizing
The active site of TBP aldolase is situated deep in the center of bonds with active site residues. DHAP P1 phosphate hydrogen
the ␣/␤8 barrel fold, as was observed for rabbit muscle aldolase. bonds with Arg278, Gly277, and Ser249; Arg278 curls around and
Continuous electron density extending from Lys205 N␨ to interacts electrostatically with the P1 oxyanion, creating a bind-
DHAP C2 shows formation of a stable covalent intermediate ing pocket, whereas Gly277 and Ser249 provide three additional
(Fig. 1A). The iminium and/or carbanion intermediate can be hydrogen bonds with the oxyanion. The intermediate is further
distinguished from the enamine intermediate because of sp2 stabilized by Gln28 hydrogen bonding DHAP O1 and its oxy-
hybridization at Lys205 N␨, as shown by the planar shape of the anion. DHAP C3 via its hydroxyl forms two hydrogen bonds
electron density observed about the Lys205 N␨. Furthermore, with Asp27 and Lys125. A water molecule Wat1, hydrogen-
DHAP O3 points slightly out of the plane defined by Lys205 N␰ bonded to Glu163, is positioned nearly perpendicular to the
and DHAP carbon atoms C1, C2, and C3, which is not possible plane of the iminium/carbanion and makes si face close contact
in the enamine intermediate and is consistent with the trapping with DHAP C3 hydroxyl (3.34 Ϯ 0.14 Å). Comparison of B-fac-
of an iminium and/or carbanion intermediate in each TBP tors of Wat1 with those of proximal water molecules suggests
21146 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 27•JULY 2, 2010

Structure of a Class I TBP Aldolase
threaded onto the native structure of S. pyogenes TBP aldolase
(see supplemental material for details). Residues Asp27, Gln28,
Arg29, Glu92, Lys125, Glu163, Lys205, Leu275, Gly277, and Arg278
,
all proximal to the DHAP intermediate in S. pyogenes TBP
aldolase, were conserved and threaded into identical positions
for all structures. The highest scoring structures identified by a
structure similarity search using the Dali server (42) were a
putative Salmonella typhimurium class I Yiht aldolase (Protein
Data Bank entry 1TO3) and rabbit muscle FBP aldolase in com-
plex with DHAP (Protein Data Bank entry 2QUT). The same
spatial disposition of residues Asp27, Gln28, Arg29, Glu92,
Lys125, Glu163, Lys205, Leu275, Gly277, and Arg278 in these struc-
tures corroborated that the S. pyogenes TBP aldolase active site
is highly conserved.
Active Site Comparison of Class I TBP and FBP Aldolases—
To investigate structural similarity of the TBP and FBP aldolase
active sites, superimposition of the DHAP-bound structures of
TBP aldolase and of FBP rabbit muscle aldolase (Protein Data
Bank entry 2QUT) (11) were performed. The best alignment
using Swiss Model (43) corresponded to a match of 148 residues
that yielded a calculated r.m.s. deviation of 1.64 Å (based on C␣
atoms) and revealed extensive homology among active site res-
idues. Finer alignment based on conserved active site residues
and DHAP atoms yielded r.m.s. deviation values of 1.06 Å (C␣
atoms) for active site residues and 0.28 Å for DHAP atoms; the
latter r.m.s. deviation value could be further decreased to 0.12 Å
when the DHAP O3 atom was excluded. Active site residues
FIGURE 2. Superposition of liganded TBP aldolase structure (cyan) with the
native enzyme structure (yellow). Three regions of continuous sequence
whose backbones undergo conformational displacement upon DHAP binding
are shown in a stick representation. For region 1, residues Asp27 and Gln28 interact
with DHAP upon binding, and in region 2, residue Ser249 interacts with DHAP,
whereas in region 3, residues Gly277 and Arg278 are shown interacting with the
carbanion intermediate. The conformational displacements narrow asymmetri-
cally the active site cavity with respect to the free enzyme.
variable occupancy by Wat1 in the four protomers. Water mol- interacting and those in close contact with the trapped covalent
ecule Wat2 hydrogen-bonds the phosphate oxyanion and intermediate, shown in Fig. 3, were largely conserved between
makes close contact with Wat1 and, contrary to Wat1, shows the two aldolases. Spatial disposition of active site residues
full occupancy in all protomers. Superposition of the four sub- Lys205, Glu163, Lys125, Asp27, Gly277, Arg278, Ser249, and Leu248
,
units of the native and DHAP-bound aldolase showed the posi- conserved in class I TBP aldolases, was identical with active site
tions of these two water molecules to be invariant to active site residues Lys229, Glu187, Lys146, Asp33, Gly302, Arg303, Ser271
,
binding.
and Leu270, conserved in class I FBP aldolases. The positions of
Conformational Changes and Active Site Binding—Compar- water molecules, Wat1 and Wat2, were similarly spatially dis-
ison of the four protomers in the asymmetric unit cell of the posed. The only striking difference was the replacement of
native TBP aldolase with those of the DHAP bound enzyme Ser300 in FBP aldolase by Leu275 in TBP aldolase. The Ser300
yielded r.m.s. deviation values of 0.98 Å using all residues and hydroxyl in the DHAP bound FBP aldolase structure forms a
0.36 Å when comparing only residues 100–230 delineating the hydrogen bond with Lys229 N␨, thus stabilizing the enamine
same central domain in each subunit that is invariant to binding intermediate (11), which is not possible by Leu275 in TBP
events. Superposition of the native and DHAP-bound struc- aldolase.
tures of TBP aldolase defined three regions that underwent sys-
Kinetic Characterization—Michaelis-Menten kinetics was
tematic conformational changes due to active site binding (Fig. used to analyze initial rate velocities measured using FBP and
2). Region 1 (residues 25–50), region 2 (residues 245–250), and TBP as substrates, and kinetic parameters kcat and Km are
region 3 (residues 275–295) contributed residues Asp27 and shown in supplemental Table S1. The data show a higher kcat
Ϫ1
Gln28, residue Ser249, and residues Gly277 and Arg278, respec- for TBP as substrate than for FBP (13 sϪ1 as opposed to 4 s
)
tively, which interacted with the covalent intermediate, thereby and a lower Km for TBP than for FBP (543 and 931 ␮M, respec-
narrowing the active site with respect to the free enzyme. The tively); substrate specificity, defined by kcat/Km, clearly shows
calculated r.m.s. deviation values between the native and ligan- preference by the enzyme for the substrate, TBP.
ded structures corresponding to the rigid body movements of
To verify the presence of carbanion in the TBP aldolase-
region 1, 2, and 3 were 0.60 Ϯ 0.36, 0.35 Ϯ 0.10, and 1.25 Ϯ 0.60 DHAP intermediate, carbanion oxidation by hexacyanofer-
Å, respectively. rate(III) was assayed and showed reduction in the presence of
Sequence Similarity—A BLAST2 search using S. pyogenes DHAP, FBP, and TBP, although turnover was lower in the pres-
TBP aldolase as a search probe yielded high sequence similarity ence of substrates (supplemental Table S1). The Michaelis
with TBP aldolases from other streptococci, S. aureus TBP kinetic parameters derived for TBP aldolase substrates in the
aldolase (5), and LacD.1, a paralogue of the LacD.2 gene that oxidation reaction were significantly smaller than for the cleav-
codes for S. pyogenes TBP aldolase, (see supplemental material). age reaction and indicated equilibration of a rate-limiting step
Three-dimensional models were built and were successfully in the carbanion oxidative shunt (supplemental Table S1).
JULY 2, 2010•VOLUME 285•NUMBER 27
JOURNAL OF BIOLOGICAL CHEMISTRY 21147

Structure of a Class I TBP Aldolase
To evaluate D2O kinetic solvent
isotope effects, enzyme activity
measurements were performed in
D2O, and resultant turnover was
compared with turnover in H2O
and is shown in supplemental Table
S1. A significant isotope effect was
observed with the largest kcat(H2O)/
k
cat(H2O) ratio of 3.3 measured for
FBP as substrate compared with 2.3
for TBP.
Proton Exchange—To determine
the rate of label exchange at DHAP
C3 hydrogen, initial rate velocities
were measured in tritiated water to
assess label uptake either as (S)-
[3-3H1]DHAP or (R)-[3-3H1]DHAP
and by the appearance of label in tri-
tiated water from (S)-[3-3H1]DHAP
or (R)-[3-3H1]DHAP. The rates of
proton exchange at C3 of DHAP
determined at saturation concen-
trations in solution and crystalline
state are shown in Table 2. TBP
aldolase labeled both pro-R and
pro-S C3 hydrogen positions of
DHAP in solution, with an approxi-
mate 2-fold kinetic preference for
pro-S labeling as compared with
pro-R. Detritiation by the enzyme
was surprising because it was unable
to detritiate label at the pro-R C3
hydrogen position of DHAP; label
release was indistinguishable from
that measured in the absence of
enzyme. A control experiment was
performed to confirm this result;
DHAP was first labeled using TBP
aldolase, and the labeled DHAP was
then detritiated again using TBP
aldolase. The amount of residual
label present as (S)-[3-3H1]DHAP
or (R)-[3-3H1]DHAP was then
measured, and rates are presented
FIGURE 3. A, superposition of the DHAP-bound TBP aldolase structure (cyan) with the DHAP bound FBP
aldolase (magenta). The superposition shows residues interacting or in close contact with DHAP, which
are conserved between the two aldolase structures. The residues Lys205, Glu163, Lys125, Asp27, Gly277
Arg278, Ser249, and Leu248 in the trapped DHAP-TBP aldolase complex superpose with residues Lys229
,
,
Glu187, Lys146, Asp33, Gly302, Arg303, Ser271, and Leu270 in the DHAP-FBP aldolase complex, respectively.
Loci of invariant water molecules, Wat1 and Wat2, in each structure were also conserved. Two notable
differences are the replacement of Glu34 and Ser300 in FBP aldolase by Gln28 and Leu275 in the TBP aldolase
structure, respectively. B, superposition of the crystal structures of native (pale cyan) and DHAP-bound
(cyan) TBP aldolase with native (pale magenta) and DHAP-bound (magenta) FBP aldolase. The regions 1, 2,
and 3, which undergo conserved conformational displacement upon ligand binding, are shown as sticks.
The overall fold of the DHAP bound TBP and FBP aldolases are shown in a schematic representation.
TABLE 2
Proton exchange of C3 hydrogen positions of DHAP
The rate of proton exchange catalyzed by TBP aldolase of the DHAP C3 hydrogen was measured by the appearance of tritium isotope label as (S)-͓3-3H1͔DHAP or
(R)-͓3-3H1͔DHAP from tritiated water (tritiation) and by the appearance of tritiated water from (S)-͓3-3H1͔DHAP and (R)-͓3-3H1͔DHAP (detritiation), as described under
“Materials and Methods.”
Tritiationa
Detritiationa
Pro-S
TBP aldolase
Totalb
Pro-Rc
Pro-Sc
Pro-R
Ϫ1
Ϫ1
s
s
Soluble enzyme
33.1 Ϯ 2.0
14.9 Ϯ 2.4
29.6 Ϯ 6.6
NDd
ND
ND
206 Ϯ 23
219 Ϯ 25
0.10 Ϯ 0.03
Control-soluble enzymee
Crystals
30.9 Ϯ 4.8
Ϫ3
Ϫ3
Ϫ3
15.0 Ϯ 2 ϫ 10
4.0 Ϯ 2 ϫ 10
13.0 Ϯ 2 ϫ 10
a Errors were calculated from at least three experiments, unless otherwise stated.
b Rate of label incorporation at (S)-͓3-3H1͔DHAP and (R)-͓3-3H1͔DHAP combined.
c Labeling of DHAP as (S)-͓3-3H1͔DHAP or (R)-͓3-3H1͔DHAP was determined using stereospecific detritiation by rabbit muscle aldolase or triose-isomerase, respectively.
d ND, not detected. Rate of detritiation was statistically insignificant relative to background.
e Detritiation of (S)-͓3-3H1͔DHAP or (R)-͓3-3H1͔DHAP previously labeled by TBP aldolase was performed using TBP aldolase.
21148 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 27•JULY 2, 2010

Structure of a Class I TBP Aldolase
in Table 2. The results show that the enzyme was not capable of aldolase and delineated amino acid residues that are responsi-
significantly detritiating pro-R-labeled DHAP although it had ble for stabilization of the covalent intermediate. Both struc-
effectively labeled the pro-R C3 position.
tural characterization and proton exchange data at C3 cor-
Exchange rates for the crystalline state shown in Table 2 were roborate the identification of the dominant steady state
considerably smaller. Label uptake in TBP aldolase crystals had intermediate as the carbanion (Fig. 1B). The carbanion is exten-
strong kinetic preference for labeling the pro-S C3 hydrogen sively stabilized by numerous electrostatic interactions and
position of DHAP. The rate of label uptake as (R)-[3-3H1]DHAP hydrogen bonds with active site residues that are nearly identi-
is not statistically different from background and indicated a cal to those observed in the enamine complex that was trapped
limit on label partitioning of Ͼ3:1 in favor of pro-S labeling in in class I FBP aldolase (Fig. 3). In addition to covalent interme-
the crystalline state. Similar to the soluble enzyme, detritiation diate formation involving an equivalent lysine residue on each
of label at the pro-R C3 hydrogen position of DHAP was not enzyme, the mode of DHAP binding in the active sites of both
detected in the crystalline enzyme because label release was enzymes is isomorphous and makes use of similar arginine, ser-
statistically indistinguishable from background. Both the rate ine, and glycine residues to grasp the P1 oxyanion while an
for labeling and detritiation at the pro-S DHAP C3 hydrogen in equivalent aspartate and lysine residues are used to bind the
the crystalline state indicate that soaking of TBP aldolase crys- DHAP C3 hydroxyl. A distinctive feature in the FBP aldolase
tals for a period of 5 min with DHAP corresponds to multiple was the preferential enamine stabilization, promoted by Ser300
turnovers in both directions and is consistent with exchange hydroxyl hydrogen-bonding Lys229 N␨ that allowed the enzyme
occurring under steady state conditions.
to differentiate between enamine and carbanion mesomers
The 7-fold greater rate of detritiation in solution compared (11). The equivalent serine residue is absent in the TBP aldolase
with tritium label incorporation in Table 2 corresponds to an active site and is replaced by Leu275. The mesomeric discrimi-
equilibrium distribution of covalent intermediates on the nation against the enamine in TBP aldolase is consistent with
enzyme where the combined enamine and carbanion popula- Leu275 destabilizing the enamine intermediate because the res-
tion exceeds that of the Schiff base intermediate by ϳ85:15. In idue cannot engage in hydrogen bond formation with Lys205
.
TBP aldolase crystals, although the uncertainties are greater,
C3 Epimerization—Class I TBP aldolases, contrary to FBP
the exchange rates indicate a similar preference (6.6-fold) for aldolases, lack a flexible C-terminal region capable of mediating
enamine and carbanion populations over the Schiff base. The si face proton transfer at C3 of the carbanion. The active site of
dominant covalent intermediate trapped on the enzyme in the TBP aldolase, however, contains two water molecules, Wat1
crystalline state is thus not an imine and is consistent with cryo- and Wat2, which are invariant to binding events (Fig. 1A) and
trapping of a predominant carbanion species in the active site. are similarly positioned with respect to bound DHAP as two
water molecules (similarly designated Wat1 and Wat2) found in
the structure of FBP aldolase (Fig. 3A). Water molecule Wat1 in
DISCUSSION
The structure determination of this class I TBP aldolase has TBP aldolase is well positioned si face to the DHAP C3 by
afforded insight into this enzyme’s mechanism and evolution. hydrogen bonding with Glu163 and is relatively mobile, whereas
The folding of the polypeptide chain of TBP aldolase, shown in water molecule Wat2 is tightly bound to the phosphate oxyan-
supplemental Fig. S1, corresponds to a (␣/␤)8 or triose-phos- ion. Wat2 is also in close contact with Wat1, making them well
phate isomerase barrel fold that is typical of class I and II situated to mediate a sequential proton transfer, as was
aldolases (44). The extensive conservation of the active site res- described for FBP aldolase (11). Such a proton exchange role is
idues and their identical spatial disposition between class I FBP not inconsistent with the observed kinetic solvent isotope effect
and TBP aldolases is consistent with the hypothesis of divergent and is supported by the virtual identity of active site residues
evolution from a common ancestor (45). The largest difference between FBP and TBP aldolases. In FBP aldolase, the P1 phos-
between the two aldolases was a loss in TBP aldolase of 23 phate dianion acts as a conjugate base to catalyze proton trans-
residues that comprise the flexible C-terminal region in FBP fer at physiological pH that is relayed through its invariant
aldolase, which interacts with the active site (11); the remaining water molecule Wat2 and activates Wat1. This activated water
differences were associated with conformations of connecting molecule, with a slight lateral shift (Ͻ1 Å) toward the DHAP C3
loops between secondary structure elements and a positional carbon in FBP aldolase, would hydrogen-bond with Lys146 and
shift in helices 5 and 6 to accommodate differential subunit position it to catalyze stereospecific pro-S ␣-proton transfer in
interfaces between the two aldolases. In TBP aldolase, the C carboxypeptidase-treated FBP aldolase (47). The ability to label
terminus position terminates at the end of helix ␣11 and is ϳ20 C3 pro-S hydrogen of DHAP by TBP aldolase would support
Å distant from its active site and therefore cannot interact with such a mechanism as water molecule, Wat1, is positioned
it. Loss of the C-terminal region in FBP aldolase reduces cleav- within close contact and si face with respect to C3. A lateral
age activity by ϳ20-fold (46) and Schiff base-carbanion inter- shift by Wat1 of ϳ1 Å enables Lys125 to hydrogen-bond Wat1
conversion by 2 orders of magnitude (47). Thus, although the and also position it for pro-S ␣-proton transfer.
two aldolases have comparable catalytic activity, the truncation
Labeling of the C3 pro-R hydrogen from the re face of DHAP
of the C-terminal region in TBP aldolase implies a divergent cannot be reconciled with a proton transfer mechanism such as
evolutionary solution to the mechanism of ␣-proton exchange that described for si face proton exchange at C3. The crystal
by the two aldolases.
structure shows no water molecule positioned roughly perpen-
DHAP Carbanion Intermediate—The trapping of the DHAP dicular and re face with respect to the plane defined by DHAP
enzymatic intermediate clearly demarked the active site in TBP C1, C2, and C3 that could mediate pro-R labeling. A water mol-
JULY 2, 2010•VOLUME 285•NUMBER 27
JOURNAL OF BIOLOGICAL CHEMISTRY 21149

Structure of a Class I TBP Aldolase
ecule positioned as the mirror image of Wat1 with respect to C3
cannot be accommodated because this would result in steric
clash with Ala25. Asp27 could act as a conjugate base to catalyze
re face proton transfer; however, from Fig. 1A, the residue is not
well positioned with respect to C3 to mediate proton transfer.
Rotations by the Asp27 side chain do not result in an orientation
favorable for re face proton transfer by Asp27. Furthermore, the
trajectory of conformational change by helical region 1 (resi-
dues 25–50), which brings Asp27 within hydrogen bonding dis-
tance of DHAP C3 hydroxyl, also does not result in a geometry
capable of pro-R labeling by Asp27 and makes this residue an
unlikely candidate for re face proton transfer.
Cis-trans Isomerization—Rotation of the hydroxymethyl
group of the bound DHAP has been postulated as a mechanism
for the apparent C3 epimerization and requires that enolization
as well as C–C bond cleavage or synthesis proceed at rates faster
than release of the products from the enzyme (48). Isotopic
studies on deoxyribose 5-phosphate aldolase have indicated
that, during the cleavage of deoxyribose 5-phosphate, rotation
of the methyl group of the enzyme-bound acetaldehyde and
enolization occurred prior to product release, resulting in an
apparent lack of stereospecificity with respect to proton uptake
at C2 (equivalent to C3 of a pyruvate lyase substrate) (48).
FIGURE 4. Isomerization of the carbanion enzymatic intermediate
formed with DHAP in the TBP aldolase active site. The DHAP C3 hydroxyl,
which is predominantly bound as the E isomer or cis configuration, is shown
modeled binding as the Z isomer or trans configuration with respect to the
Indeed, proton exchanges in TBP aldolase, shown in Table 2, DHAP C2-Lys205 N␨ bond. The Z isomer results in formation of two hydrogen
bonds (shown in orange) by DHAP C3 hydroxyl with DHAP O1 and Gln28 and
appear to proceed at rates faster than C–C bond cleavage. Rota-
would allow protonation of the pro-R position of DHAP C3 by the same ste-
reofacial proton transfer mechanism, implicating water molecules Wat1 and
tion about the equivalent C2–C3 bond in the DHAP interme-
Wat2 as used for protonation of the pro-S position of DHAP C3 in Figs. 1A and
diate of TBP and FBP aldolases is, however, restricted due to its
3A. Gln28 is positioned by its two hydrogen bonds made with the DHAP ester
double bond character and limits the rotamers to the two dis-
oxygen and oxyanion. Pertinent hydrogen bonds or close contacts present in
the E isomer are shown in green. Such geometry is feasible for sp2 hybridiza-
crete isomers: E, where the DHAP O3 hydroxyl is oriented cis to
tion at C3 in the carbanion, whereas sp3 hybridization in the Schiff base or
the DHAP C2–Lys205 N␨ bond, and Z, where the orientation is
iminium intermediate would result in an unacceptable close contact
between DHAP C3 hydroxyl and DHAP O1.
trans. Their relative populations depend on the active site sta-
bilization of each isomer, and their rate of interconversion is
determined by the height of the rotational barrier, which ization. Together, these considerations explain the inability to
depends on the extent of double bond character at C2-C3. The detect pro-R labeling of the DHAP C3 hydrogen in FBP aldolase
rotational barrier would be greater in the enamine than in the (16). The loss of stereospecific labeling at C3 in TBP aldolase is
carbanion, which has reduced C2-C3 double character.
thus a likely consequence of cis-trans isomerization of the car-
Fig. 1, A and B, shows the DHAP intermediate bound as the E banion promoted by Gln28 and Leu275. From an evolutionary
isomer, whereas Fig. 4 shows a possible conformation for the Z perspective, C3 epimerization in class I DHAP-dependent aldo-
isomer as modeled from the E isomer. Stabilization of the lases would be based on a conserved stereospecific proton
DHAP C3 hydroxyl in the E isomer implicates three hydrogen transfer mechanism exploiting cis-trans isomerization in the
bonds with active residues Asp27 and Lys125. The DHAP C3 carbanion intermediate.
hydroxyl, by adopting a trans configuration in Fig. 4, forms a
The preferential labeling in the crystalline state of the pro-S
hydrogen bond with DHAP O1 (2.4–2.6 Å) that would be fur- C3 hydrogen with respect to the pro-R DHAP C3 hydrogen of
ther stabilized by hydrogen bonding with Gln28. The observed Ͼ3:1 implies a population for the Z isomer that would have
2:1 ratio in the rates of pro-S and pro-R labeling at C3 of DHAP occupancy of less than 25% in the crystal structure and which
in TBP aldolase is consistent with a mechanism of stereofacial si would make it technically challenging to observe above the
face proton transfer at C3 in both E and Z isomers with more noise level in the electron density map. The small exchange
extensive hydrogen bonding stabilizing the E isomer. Preferen- rates in the crystalline state as well as the labeling pattern would
tial stabilization of carbanion mesomer by the enzyme would argue that crystal lattice packing decreases exchange rates and
favor the requisite cis-trans isomerization about the C2–C3 differentially stabilizes Z and E isomer populations with respect
bond due to its reduced double bond character in the carban- to solution. Indeed, in all subunits, conformational mobile
ion. In FBP aldolase, the presence of Glu34, equivalent to Gln28 regions 1 and 2 that respond to DHAP binding participate in
in TBP aldolase (Fig. 3A), would not favor hydrogen bonding crystal packing contacts and thus could redirect Z and E isomer
with DHAP O3 hydroxyl in the Z isomer compared with TBP stability and the barrier height of cis-trans isomerization. Fluc-
aldolase, because such an interaction with Glu34 would simul- tuations along the conformational trajectory made by region 1
taneously make a repulsive interaction with DHAP O1. Fur- upon DHAP binding, which could disrupt Asp27 hydrogen
thermore, the greater double bond character of the C2–C3 bond formation with DHAP O3 and facilitate cis-trans isomer-
bond in the enamine heightens the barrier to cis-trans isomer- ization, also would be hindered by the crystal lattice packing.
21150 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 27•JULY 2, 2010

Structure of a Class I TBP Aldolase
The Z isomer configuration shown in Fig. 4 is however only tional changes in S. pyogenes would represent a novel target to
possible if the carbanion intermediate has sp2 hybridization at inhibit virulence of this versatile human pathogen, which is the
C3 because sp3 hybridization at C3 would result in an unaccept- causative agent of numerous human diseases, ranging from
able close contact between DHAP C3 hydroxyl and DHAP O1 pharyngitis and impetigo to the often fatal necrotizing fasciitis
in this isomer. A planar carbanion center is favored because the and septicemia (51). The substrate ambiguity benefits the sim-
nucleophile is adjacent to the DHAP C2- Lys205 N␨ double ple fermentative energy metabolism of S. pyogenes by allowing
bond that affords conjugative stabilization. The destabilization it to respond and adapt to environmental alterations in carbon
of the Z isomer in the Schiff base, due to its sp3 hybridization at sources (49).
C3, would be consistent with a very low rate of pro-R detritia-
Acknowledgments—Work was carried out in part at beamlines X12B,
X25, and X29 of the National Synchrotron Light Source (supported
principally by the Offices of Biological and Environmental Research
and of Basic Energy Sciences of the United States Department of
Energy and the National Center for Research Resources of the
National Institutes of Health). Assistance by beamline personnel, Drs.
L. Flaks, D. K. Schneider, A. Soares, A. He´roux, and H. Robinson, is
gratefully acknowledged. The help of Laurent Cappadoccia in data
collection was particularly appreciated. Critical reading of the manu-
script by Dr. Casimir Blonski was also appreciated. Tagatose 1,6-
bisphosphate was a generous gift of Dr. W. Fessner.
tion at C3, which was not detected, in comparison with a very
high rate of pro-S detritiation at C3, as shown in Table 2. The
inhibition of the Schiff base to carbanion interconversion and a
kinetic preference for pro-S labeling indicate a transaldolase
rather than aldolase activity toward sorbose bisphosphate and
psicose bisphosphate as substrates.
Induced Conformational Changes—In Fig. 2, three regions in
the native enzyme are shown to undergo small yet significant
conformational changes upon active site binding by DHAP.
The conformational changes consist of rigid body movements
induced by DHAP that serve to stabilize the DHAP-enzyme
intermediate and are very akin to those observed in the struc-
tures of rabbit muscle aldolase bound to DHAP and FBP (11, REFERENCES
27). Both FBP and TBP aldolases respond to active site binding
by identical asymmetrical active site narrowing, implicating
equivalent ␣-helices and regions of the ␤-turn flanking the
active site, as shown in Fig. 3B. Conformational displacements
by these secondary structure features serve in both cases to bind
the DHAP phosphate oxyanion and C3 hydroxyl. The low
sequence homology between those two class I aldolases that are
functionally very similar suggests not only that the ␣/␤ barrel
platform is functionally robust and able to tolerate considerable
sequence divergence but also that the conformational changes
requisite to binding are strongly conserved, being invariant to
the same degree of sequence divergence.
1. Suri, J. T., Mitsumori, S., Albertshofer, K., Tanaka, F., and Barbas, C. F., 3rd
(2006) J. Org. Chem. 71, 3822–3828
2. Takayama, S., McGarvey, G. J., and Wong, C. H. (1997) Annu. Rev. Micro-
biol. 51, 285–310
3. Fessner, W. D. (1998) Curr. Opin. Chem. Biol. 2, 85–97
4. Wong, C. H., Whitesides, G. M. (1994) Enzymes in Synthetic Organic
Chemistry, pp. 195–232, 1st Ed., Elsevier Science Publishing Co., Inc.,
New York
5. Bissett, D. L., and Anderson, R. L. (1980) J. Biol. Chem. 255, 8750–8755
6. Lee, J. H., Bae, J., Kim, D., Choi, Y., Im, Y. J., Koh, S., Kim, J. S., Kim, M. K.,
Kang, G. B., Hong, S. I., Lee, D. S., and Eom, S. H. (2006) Biochem. Biophys.
Res. Commun. 347, 616–625
7. Grazi, E., Rowley, P. T., Cheng, T., Tchola, O., and Horecker, B. L. (1962)
Biochem. Biophys. Res. Commun. 9, 38–43
The conformational changes observed in TBP aldolase may
thus represent the molecular basis enabling the LacD.1 para-
logue of TBP aldolase in S. pyogenes and to which TBP aldolase
is highly homologous (73% identical and 86% similar) to act as a
putative metabolic sensor that regulates global transcription of
virulence genes in S. pyogenes (49, 50). Sequence differences
between the two paralogues are largely at the level of surface-
exposed residues whereas active site residues are conserved and
implicitly support the hypothesis of homologous conforma-
tional changes in LacD.1 triggered by active site binding. All
residues in S. pyogenes LacD.2 found proximal to the bound
8. Kobashi, K., Lai, C. Y., and Horecker, B. L. (1966) Arch. Biochem. Biophys.
117, 437–444
9. Kobes, R. D., Simpson, R. T., Vallee, R. L., and Rutter, W. J. (1969) Bio-
chemistry 8, 585–588
10. Harris, C. E., Kobes, R. D., Teller, D. C., and Rutter, W. J. (1969) Biochem-
istry 8, 2442–2454
11. St-Jean, M., and Sygusch, J. (2007) J. Biol. Chem. 282, 31028–31037
12. Rose, I. A. (1969) Comprehensive Biochemistry, pp. 93–161, Vol. 17
(Florkin, M., and Stoltz, E. H., eds) Elsevier Science Publishing Co.,
Inc., New York
13. Jencks, W. P. (1987), Catalysis in Chemistry and Enzymology, pp.
120–127, Courier Dover Publications, New York
14. Grazi, E., Cheng, T., and Horecker, B. L. (1962) Biochem. Biophys. Res.
Commun. 7, 250–253
DHAP intermediate (namely Asp27, Gln28, Arg29, Glu92, Lys125
,
Glu163, Lys205, Leu275, Gly277, and Arg278) are sequence-con-
served and spatially conserved in the computed LacD.1 model.
Furthermore, residues vicinal to the active site were also con-
served both in identity and position. Experimental data indicate
that the role of LacD.1 depends on substrate recognition, espe-
cially on DHAP, but not on enzymatic activity and is most sen-
sitive to changes in DHAP levels (49). Under conditions of high
glycolysis, product binding by DHAP could thus induce a con-
formational change in LacD.1. Indirect evidence suggests that
LacD.1 sequesters a regulatory partner and, by inhibiting it
from activating its target genes, triggers virulence gene expres-
15. Rose, I. A., and Rieder, S. V. (1958) J. Biol. Chem. 231, 315–329
16. Rose, I. A. (1958) J. Am. Chem. Soc. 80, 5835–5836
17. Chiu, T. H., and Feingold, D. S. (1965) Biochem. Biophys. Res. Commun.
19, 511–516
18. Chiu, T. H., and Feingold, D. S. (1969) Biochemistry 8, 98–108
19. Kobes, R. D., and Dekker, K. E. (1969) J. Biol. Chem. 244, 1919–1925
20. Meloche, H. P., and Mehler, L. (1973) J. Biol. Chem. 248, 6333–6338
21. Espelt, L., Bujons, J., Parella, T., Calveras, J., Joglar, J., Delgado, A., and
Clape´s, P. (2005) Chemistry 11, 1392–1401
22. Zgiby, S. M., Thomson, G. J., Qamar, S., and Berry, A. (2000) Eur. J. Bio-
chem. 267, 1858–1868
23. Williams, G. J., Domann, S., Nelson, A., and Berry, A. (2003) Proc. Natl.
Acad. Sci. U.S.A. 100, 3143–3148
sion (49). Interference with or exploitation of these conforma- 24. Sygusch, J., Beaudry, D., and Allaire, M. (1987) Proc. Natl. Acad. Sci. U.S.A.
JULY 2, 2010•VOLUME 285•NUMBER 27
JOURNAL OF BIOLOGICAL CHEMISTRY 21151

Structure of a Class I TBP Aldolase
84, 7846–7850
37. Schu¨ttelkopf, A. W., and van Aalten, D. M. (2004) Acta Crystallogr. D Biol.
Crystallogr. 60, 1355–1363
25. Chudzik, D. M., Michels, P. A., de Walque, S., and Hol, W. G. (2000) J. Mol.
Biol. 300, 697–707
38. Weiss, M. (2001) J. Appl. Crystallogr. 34, 130–135
39. Collaborative Computational Project 4 (1994) Acta Crystallogr. D 50,
760–763
26. Dalby, A. R., Tolan, D. R., and Littlechild, J. A. (2001) Acta Crystallogr. D
Biol. Crystallogr. 57, 1526–1533
27. St-Jean, M., Lafrance-Vanasse, J., Liotard, B., and Sygusch, J. (2005) J. Biol.
Chem. 280, 27262–27270
40. Diamond, R. (1992) Protein Sci. 1, 1279–1287
41. DeLano, W. L. (2002) The PyMOL Molecular Graphics System, DeLano
Scientific LLC, San Carlos, CA
28. Liotard, B., and Sygusch, J. (2004) Acta Crystallogr. D 60, 528–530
29. Racker, E. (1947) J. Biol. Chem. 167, 843–854
42. Holm, L., Ka¨a¨ria¨inen, S., Rosenstro¨m, P., and Schenkel, A. (2008) Bioin-
formatics 24, 2780–2781
30. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307–326
31. Terwilliger, T. C., and Berendzen, J. (1999) Acta Crystallogr. D 55,
849–861
43. Guex, N., and Peitsch, M. C. (1997) Electrophoresis 18, 2714–2723
44. Lorentzen, E., Siebers, B., Hensel, R., and Pohl, E. (2004) Biochem. Soc.
Trans. 32, 259–263
32. Terwilliger, T. C. (2000) Acta Crystallogr. D 56, 965–972
33. Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta
Crystallogr. A. 47, 110–119
45. Wierenga, R. K. (2001) FEBS Lett. 492, 193–198
46. Spolter, P. D., Adelman, R. C., and Weinhouse, S. (1965) J. Biol. Chem. 240,
1327–1337
34. Bru¨nger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P.,
Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S.,
Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta
Crystallogr. D 54, 905–921
47. Rutter, W. J., Richards, O. C., and Woodfin, B. M. (1961) J. Biol. Chem.
236, 3193–3197
48. Corina, D. L., and Wilton, D. C. (1976) Biochem. J. 157, 573–576
49. Loughman, J. A., and Caparon, M. G. (2007) Mol. Microbiol. 64, 269–280
50. Loughman, J. A., and Caparon, M. G. (2006) EMBO J. 25, 5414–5422
51. Cunningham, M. W. (2000) Clin. Microbiol. Rev. 13, 470–511
35. Adams, P. D., Grosse-Kunstleve, R. W., Hung, L. W., Ioerger, T. R., Mc-
Coy, A. J., Moriarty, N. W., Read, R. J., Sacchettini, J. C., Sauter, N. K., and
Terwilliger, T. C. (2002) Acta Crystallogr. D 58, 1948–1954
36. Emsley, P., and Cowtan, K. (2004) Acta Crystallogr. D 60, 2126–2132
21152 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 27•JULY 2, 2010

Enzymology:
Structure of a Class I
Tagatose-1,6-bisphosphate Aldolase:
INVESTIGATION INTO AN APPARENT
LOSS OF STEREOSPECIFICITY
Clotilde LowKam, Brigitte Liotard and Jurgen
Sygusch
J. Biol. Chem. 2010, 285:21143-21152.
doi: 10.1074/jbc.M109.080358 originally published online April 28, 2010
Access the most updated version of this article at doi: 10.1074/jbc.M109.080358
Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites.
Alerts:
• When this article is cited
• When a correction for this article is posted
Click here to choose from all of JBC's e-mail alerts
Supplemental material:
http://www.jbc.org/content/suppl/2010/04/28/M109.080358.DC1.html
This article cites 48 references, 14 of which can be accessed free at
http://www.jbc.org/content/285/27/21143.full.html#ref-list-1