CH-π Interactions Promote the Conversion of Hydroxypyruvate in a Class II Pyruvate Aldolase

Article abstract of DOI:10.1002/adsc.201900205

The class II hydroxy ketoacid aldolase A5VH82 from Sphingomonas wittichii RW1 (SwHKA) accepts hydroxypyruvate as nucleophilic donor substrate, giving access to synthetically challenging 3,4-dihydroxy-α-ketoacids. The crystal structure of holo-SwHKA in complex with hydroxypyruvate revealed CH-π interactions between the C?H bonds at C3 of hydroxypyruvate and a phenylalanine residue at position 210, which in this case occupies the position of a conserved leucine residue. Mutagenesis to tyrosine further increased the electron density of the interacting aromatic system and effected a rate enhancement by twofold. While the leucine variant efficiently catalyses the enolisation of hydroxypyruvate as the first step in the aldol reaction, the enol intermediate then becomes trapped in a disfavoured configuration that considerably hinders subsequent C?C bond formation. In SwHKA, micromolar concentrations of inorganic phosphate increase the catalytic rate constant of enolisation by two orders of magnitude. This rate enhancement was now shown to be functionally conserved across the structurally distinct (α/β)₈ barrel and αββα sandwich folds of two pyruvate aldolases. Characterisation of the manganese (II) cofactor by electron paramagnetic resonance excluded ionic interactions between the metal centre and phosphate. Instead, histidine 44 was shown to be primarily responsible for the binding of phosphate in the micromolar range and the observed rate enhancement in SwHKA. (Figure presented.).

Full text of DOI:10.1002/adsc.201900205

Title: CH-π Interactions Promote the Conversion of Hydroxypyruvate in
a Class II Pyruvate Aldolase
Authors: Stefan Marsden, Luuk Mestrom, Isabel Bento, Duncan
McMillan, Peter-Leon Hagedoorn, and Ulf Hanefeld
This manuscript has been accepted after peer review and appears as an
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To be cited as: Adv. Synth. Catal. 10.1002/adsc.201900205
Link to VoR: http://dx.doi.org/10.1002/adsc.201900205

Advanced Synthesis & Catalysis
10.1002/adsc.201900205
FULL PAPER
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
CH-π Interactions Promote the Conversion of
Hydroxypyruvate in a Class II Pyruvate Aldolase
[
a,b]
[a]
[b]
Stefan R. Marsden , Luuk Mestrom , Isabel Bento* , Peter-Leon
[
a]
[a]
[a]
Hagedoorn , Duncan G.G. McMillan and Ulf Hanefeld*
a
Biokatalyse, Afdeling Biotechnologie, Technische Universiteit Delft, van der Maasweg 9, 2629HZ Delft,
The Netherlands. U.hanefeld@tudelft.nl, +31 15 278 9304
EMBL Hamburg, Notkestrasse 85, 22607 Hamburg, Germany. ibento@embl-hamburg.de
b
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.201######.((Please delete if not appropriate))
Abstract. The class II hydroxy ketoacid aldolase A5VH82 considerably hinders subsequent C-C bond formation. In
from Sphingomonas wittichii RW1 (SwHKA) accepts SwHKA, micromolar concentrations of inorganic phosphate
hydroxypyruvate as nucleophilic donor substrate, giving increase the catalytic rate constant of enolisation by two
access to synthetically challenging 3,4-dihydroxy-α- orders of magnitude. This rate enhancement was now
ketoacids. The crystal structure of holo-SwHKA in complex shown to be functionally conserved across the structurally
with hydroxypyruvate revealed CH-π interactions between distinct (α/β)
8
barrel and αββα sandwich folds of two
the C-H bonds at C3 of hydroxypyruvate and pyruvate aldolases. Characterisation of the manganese (II)
a
phenylalanine residue at position 210, which in this case cofactor by electron paramagnetic resonance excluded ionic
occupies the position of a conserved leucine residue. interactions between the metal centre and phosphate.
Mutagenesis to tyrosine further increased the electron Instead, histidine 44 was shown to be primarily responsible
density of the interacting aromatic system and effected a rate for the binding of phosphate in the micromolar range and
enhancement by twofold. While the leucine variant the observed rate enhancement in SwHKA.
efficiently catalyses the enolisation of hydroxypyruvate as
the first step in the aldol reaction, the enol intermediate then Keywords:
pyruvate;
hydroxypyruvate;
aldolase;
becomes trapped in a disfavoured configuration that
CH-π interactions; phosphate activation
extensive screening of 571 aldolases, of which only
19 enzymes showed an observable promiscuity
towards hydroxypyruvate (HPA) as nucleophilic
Introduction
Aldolase catalysed asymmetric C-C bond donor substrate.[10] In particular, aldolase A5VH82
formation plays a pivotal role in the synthesis of from Sphingomonas wittichii RW1 showed good
[1]
[2]
many chiral building blocks, natural compounds,
conversions, giving new access to several
pharmaceuticals[ and has been industrially applied 3,4-dihydroxy-α-ketoacids by variation of the
on a multi ton scale.[ Due to this wide-ranging acceptor aldehyde. Intrigued by its unusual activity,
utility, its relevance for synthetic chemistry is well we set out to elucidate the molecular basis behind the
3]
4]
[5]
recognized. There are two distinct types of aldolases enzyme’s widened donor substrate scope and to
that are classed by their respective mode of action. conduct an extensive biochemical and structural
Class I aldolases function via a Schiff base characterisation. Our findings motivated us to denote
mechanism involving a lysine residue, while class II this enzyme as a hydroxy ketoacid aldolase (SwHKA,
aldolases require a divalent metal cation as cofactor scheme 1).
for substrate binding and activation.[7] In general,
[6]
aldol reactions are fully reversible via the same
reaction pathway. In nature, such retro-aldol reactions
are found in biodegradation pathways of aromatic
compounds such as phenols, biphenyls and lignin
[8]
derived metabolites, which then can be metabolised.
For synthetic purposes, aldolase catalysed reactions
with pyruvate are well established and give
[1a, 9]
straightforward access to 4-hydroxy-α-ketoacids.
Scheme 1. SwHKA catalysed aldol coupling of
Unfortunately, wild type pyruvate aldolases typically hydroxypyruvate with an acceptor aldehyde to afford a
[
10]
show a strict requirement for a single donor substrate, 3(S),4(S)-dihydroxy-α-ketoacid.
which considerably limits their product scope. To
address this issue, de Berardinis et al. conducted an
1
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Advanced Synthesis & Catalysis
10.1002/adsc.201900205
2
+
A first indication of phosphate being involved in the Crystal structure of Mg -SwHKA (6r62.pdb)
catalytic mechanism of class II pyruvate aldolases
was given by the crystal structure of the The crystal structure of holo-SwHKA in complex with
2
-dehydro-3-deoxy-galactarate
aldolase
from hydroxypyruvate was determined at 1.55
Å
Escherichia coli (DDG, 1dxf.pdb). Based on the resolution. The final structural model corresponds to a
apparent absence of catalytically active amino acid monomeric subunit (figure S1) and includes all 251
residues and an increased activity in phosphate buffer, amino acid residues plus one histidine residue from
2
+
the authors proposed a reaction mechanism which the His
6
-tag. Additionally, one Mg ion and one
hydroxypyruvate moiety were found fully occupied in
This hypothesis has since been challenged by the electron-density maps, together with 352 solvent
observations for the homologous enzyme HpaI (44% molecules. The monomeric subunit shows an (/)
sequence identity with DDG) where lower activities triosephosphate isomerase (TIM) fold, similar to the
[11]
involved inorganic phosphate as the catalytic base.
8
[12]
were found in the presence of phosphate. While a structures of the homologues 4-hydroxy-2-oxo-
[15]
recent study on the class II HMG/CHA pyruvate heptane-1,7-dioate aldolase (HpaI) and 2-dehydro-
aldolase described a 10-fold rate enhancement by 3-deoxygalactarate aldolase (DDG).[
11]
The
8
th
inorganic phosphate, these findings were attributed to -helix extends out of the -barrel and packs onto a
the enzyme’s distinct structure (αββα sandwich fold -sheet of a neighbouring two-fold related subunit to
[13]
in the HMG/CHA aldolase
vs. an (α/β) barrel fold form a domain-swapped dimer (figure S2). In
8
[11]
in the DDG aldolase ; 16.8% shared sequence agreement with previous results from size exclusion
identity).[ Here we present new insights into the chromatography, a final hexameric assembly was
mode of phosphate activation by identifying the key obtained by application of the 3-fold crystallographic
residues that are involved in the binding of phosphate symmetry to this domain swapped dimer. (figure S3,
and the concomitant rate enhancement. Our results S4). Comparison with the homologous enzymes HpaI
show that phosphate activation is functionally (rmsd 1.54 Å for 243/251 aligned residues) and DDG
conserved within the same order of magnitude in the (rmsd 1.61Å for 243/251 aligned residues) indicated,
14]
(
α/β) barrel fold of SwHKA and thereby not a unique that the overall fold is conserved in SwHKA. The
8
property of the αββα sandwich fold. Furthermore, active site is located inside a cavity close to the
using the crystal structure of holo-SwHKA in C-terminus, and comprises of residues from two
complex with hydroxypyruvate, we were able to different monomeric subunits and a metal cluster
2
+
demonstrate CH-π interactions between residue F210 (figure 1). The Mg ion is coordinated by two
and HPA to be responsible for its conversion. Based carboxylate groups from residues E145 and D171 in a
on this, we could then rationally devise a variant of monodentate fashion, two water molecules (W1, W2)
two-fold improved turnover rate.
and the bidentate binding of hydroxypyruvate via its
carbonyl and the carboxylate group completes the
octahedral geometry.
Results and Discussion
Biocatalyst production
The SwHKA gene was codon optimised for
recombinant expression in E. coli BL21(DE3) and a
purified protein yield of up to 75 mg/L medium was
obtained upon single-step metal affinity purification
via its N-terminal His
in E. coli BL21Star(DE3) yielded approximately
.5 g/L of insoluble inclusion bodies with SwHKA as
6
-tag. Notably, gene expression
2
its main constituent, while only 10 - 20 mg/L was
present in its soluble form. The inclusion bodies were
purified with sodium deoxycholate and subsequently
re-dissolved in urea, but no functional enzyme could
be recovered. Cheap and high yielding biocatalyst
production from purified inclusion bodies was
therefore not possible. Overall, gene expression in
E. coli BL21(DE3) gave higher yields of soluble
protein. For crystallisation studies, the soluble
enzyme was further purified by size exclusion
chromatography resulting in
a
single peak
corresponding to a hexameric homo-oligomer (27.4
kDa x 6 = 164.4 kDa), a commonly found structural
Figure 1. Representation of the relevant active site residues
in Mg -SwHKA (6r62.pdb). The backbones of the two
2+
[11, 15]
state of other class II pyruvate aldolases.
monomeric subunits that form the active site are coloured
[
16]
in grey and yellow. Figures were created with PyMol.
2
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Advanced Synthesis & Catalysis
10.1002/adsc.201900205
HPA therefore coordinates the metal cofactor in a for the increased Lewis acidity, where the connecting
[
11,
similar way as pyruvate in homologous structures.
line between the two tops of the square-based
1
5]
[20]
The carboxylate group in HPA is located within pyramid deviates from the expected angle of 180°.
hydrogen bond distance to the backbone NH and side A similar deviation was also observed in the crystal
chain hydroxyl of T170. The carbonyl group in HPA structure of holo-SwHKA for the Mg2+ cluster
forms a hydrogen bond with R69 and its C3 hydroxyl (6r62.pdb).
group is within hydrogen bond distance of the
carbonyl backbone of I167. Furthermore, a well-
ordered water molecule is observed at 3 Å distance
from C3 in HPA. This water molecule constitutes a
good candidate for the catalytic deprotonation at C3,
similar to the proposed role of water in the structure
of pyruvate bound HpaI (4b5u.pdb).[15] This bridging
interaction is necessary due to the absence of
proximal amino acid residues for
a
direct
deprotonation. Notably, F210 replaces a conserved
leucine residue (L212 and L216 for HpaI and DDG,
respectively) as the most apparent difference in active
site geometries. The C3 C-H bonds in HPA are
oriented towards the aromatic system of F210 and
establish CH-π interactions.[17] This hydrophobic
region was previously proposed to flank the aldehyde
binding locus.[ Another important and conserved
residue in the active site is H44 (H45 and H50 in
HpaI and DDG, respectively). H44 is located within
hydrogen bond distance of D83’ in the symmetry
15]
Figure 2. Metal dependent relative activities of SwHKA
related subunit and of a water molecule that
2+
coordinates the Mg2+ ion. Together with the loop
under metal saturated conditions (10 mM M , 20 mM
TEOA, pH 8.0). Activities were determined for the
retro-aldol type decarboxylation of oxaloacetate by a
NADH/LDH coupled enzyme assay and the results were
region that comprises of residues 115’ to 120’ in the
symmetry related molecule, D83’ is involved in a
hydrogen bond network that contributes to the
integrity of the active site. H44 forms hydrogen bonds
with R120’ and D171. Furthermore, D171 forms
2+
normalized for Mn .
To determine the pH optimum, the enzyme activity
was measured in several buffer systems over a range
from 5.5 to 8.25 and the optimum was found at pH
hydrogen bonds with F117’, S116’ and one of the
water molecules from the Mg2+ coordination sphere.
Although this loop shows low sequence conservation
among the homologous enzymes, it maintains the
active site configuration via the hydrogen bond
network.
6
.75. Notably, long-term storage of the enzyme at
-
20°C required alkaline conditions (pH ≥7.5), under
which the enzyme retained full activity for more than
one month (data not shown). Incubation in potassium
phosphate buffer substantially increased the reaction
rate in comparison with other buffer salts. Analysis
over a broad range of concentrations revealed a
considerable affinity towards phosphate, with an
apparent dissociation constant of Kd,Pi = 175±36µM.
This observation was of particular interest, since
phosphate buffers were reported to exhibit an adverse
effect on the activity of class II aldolases in several
Biochemical characterisation
In class II aldolases, divalent metal ions play a central
[7]
role in catalysis. For SwHKA, a slight preference
2
+
2+
2+
for Mn over Mg and Co was previously reported,
but no dissociation constants or kinetic parameters
were measured.[
activity of the enzyme was screened, including Ni ,
10]
Therefore, the metal dependent
2
+
[12, 21]
[14]
2
+
2+
other cases,
with only one exception.
Ca and Zn , with the latter being commonly found
in the dihydroxyacetonephosphate specific subgroup
Strikingly, even minor concentrations of phosphate
significantly increased the reaction rate up to 13-fold
under standard assay conditions, from 2.1 U/mg to
[18]
of class II aldolases.
The highest activity was
observed with Mn2 as cofactor, followed by Mg
+
2+
2+
v
sat = 27.1 U/mg following saturation kinetics with
with 83% of the activity of Mn (figure 2). While
2
+
respect to phosphate (figure 3). The metal dissociation
rather similar in rate, a strong preference for Mn
2
+
2+
2
+
constants for Mn and Mg were unaffected by the
presence/absence of phosphate, rendering a direct
ionic interaction unlikely (table 1). Henceforth, we
directed our attention towards the two positively
charged active site residues R69 and H44, which
appeared to be likely candidates involved in
phosphate binding. While H44 also plays a structural
role by interacting with D83 from another dimer,
over Mg was observed with respect to their apparent
2+
binding constants, with a K
d
of 3.3±0.6 µM for Mn
2+
in contrast to 58.4±4.1 µM for Mg . These results are
2+
2+
of particular interest, since solvated Mg and Mn
ions are known to be poor Lewis acid catalysts in
aqueous media.[
19]
2+
A previous study on the Mn
containing enzyme GtHNL suggested a distorted
octahedral coordination geometry to be responsible
3
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Advanced Synthesis & Catalysis
10.1002/adsc.201900205
mutant
H44A
still
displayed
40% phosphate is not essential for catalysis. These results
(
0.78±0.01 U/mg) residual activity in the absence of are consistent with previous studies, where a
phosphate.
structural molecule of water was identified as the
catalytic base, due to the lack of proximal amino acid
residues for a direct deprotonation at C3 in the
[9a,
12,
15,
22]
substrate.
Ultimately, elevated
concentrations of phosphate (>10 mM KPi) were
found to have a negative impact on the reaction rate
(
figure S23). We propose this to be due to the
typically low solubility of divalent metal phosphates,
which deprives the active site from its metal
[23]
cofactor. Notably, a higher rate of reaction can still
be observed for states where the apoenzyme
predominates, due to the rate enhancing effect of
phosphate. This issue was addressed experimentally
by using the minimal concentrations of metal and
phosphate that are required for establishing saturating
conditions. These findings plausibly clarify the
previous misconception of the effect of phosphate in
literature, where it typically was added in a large
excess of 50 - 1000 molar equivalents with respect to
[
12, 21, 22d]
the metal.
Finally, we would like to mention
Figure 3. Catalytic rate enhancement in SwHKA by the
that amine buffers are known organocatalysts for the
transketolase-like conversion of hydroxypyruvate.
presence inorganic phosphate under metal saturated
[24]
2
+
2+
conditions (0.1 mM Mn , 1 mM Mg , 5 mM TEOA, pH
.5). Rates were determined by the coupled enzyme assay.
Using decarboxylation as driving force, this reaction
then affords a racemic dihydroxyketone in place of
7
[25]
the desired aldol product. This considerably limits
the range of buffer systems which would allow for
selective aldol reactions with hydroxypyruvate at
neutral pH. In theory, structural analogues of
phosphate with matching pKa values should also be
However, the mutant’s affinity towards phosphate
was reduced 36-fold with Kd,Pi = 6.22±0.53 mM and
the related rate enhancement was only 3-fold
(
2.23±0.08 U/mg, figure S22). Since both phosphate
binding and activation are not completely lost by a
replacement with alanine, H44 seems to be primarily,
but not solely responsible for the observed activation
of SwHKA by inorganic phosphate. In agreement with
previous studies, a complete loss of activity was
observed for the R69A mutant both in the absence
and presence of saturating concentrations of
phosphate. These findings demonstrate, that neither
3
-
able to act as the catalytic base. Indeed, AsO
4
and
3-
VO
HMG/CHA aldolase, while SO
4
were previously shown to similarly activate the
2
-
2-
4
and MoO
4
did
[14]
not. A brief overview of phosphate analogues and
their corresponding pKa values is given in table S3.
EPR characterisation of the metal centre
H44 in combination with P , nor phosphate alone can
act as the catalytic base and that R69 is required.
i
To further elucidate the interplay between the metal
cofactor, inorganic phosphate and the substrates, the
2+
metal centre was analysed by EPR (figure 4). Mn is
paramagnetic, characterised by high spin
S = 5/2 and the hyperfine coupling of the nuclear spin
Table 1. Overview of apparent metal / phosphate
dissociation constants and the corresponding saturation
kinetics vsat under standard assay conditions (0.5 mM
oxaloacetate).
a
5
5
I = 5/2 of Mn (100% natural abundance) and was
therefore chosen as the metal cofactor for EPR
Enzyme M2+ Pi
v
sat (U/mg)
K
K
2+
d,M2+
d,Pi
(mM)
experiments. The Mn EPR is characteristic, as it is a
(
µM)
3.3±0.6
1.65±0.03 58.4±4.1
27.0±0.67
17.21±0.29 158±12 0.13±0.02
unique case where the zero-field splitting (which is
independent of the magnetic field) is smaller or equal
to the Zeeman interaction (dependent on the magnetic
field). This is contrary to most other paramagnetic
metal ions, for which the zero field splitting is much
2
2
2
2
2
2
+
+
+
+
+
+
WT
WT
WT
WT
H44A
H44A
Mn
Mg
Mn
Mg
Mn
Mn
-
-
+
+
-
2.15±0.09
-
-
6.5±0.7 0.18±0.04
a)
0.78±0.01
2.23±0.08
n.d.
n.d.
-
3+
larger, e.g. high spin Fe (S = 5/2). The spectrum is
a)
+
6.22±0.53
very broad because of five allowed transitions with
each Δm = 1, and each of these transitions is split
s
a)
5
5
n.d. = not determined
into six lines due to the Mn hyperfine coupling.
2
+
Normally, Mn is dominated by the m
s
= -1/2 to
m
s
= +1/2 transition, which results in a broad signal
Despite a low dissociation constant, measurements of
inorganic phosphate after metal affinity
chromatography did not support its enzyme bound
co-purification, indicating that while beneficial,
around g = 2 with the characteristic splitting into six
lines. Additional lines result from semi-forbidden
transitions that have a lower intensity. A more
2
+
extensive description of Mn EPR of proteins has
4
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Advanced Synthesis & Catalysis
10.1002/adsc.201900205
[20]
been published previously. The EPR spectrum of ion, which would result from a change in the
2
+
Mn
bound aldolase is dominated by the coordination number and geometry. The change after
characteristic six line pattern around g = 2, with a addition of hydroxypyruvate is subtler, but significant
hyperfine coupling constant of circa 95 Gauss. This and in agreement with the bidentate coordination of
hyperfine coupling constant is consistent with an hydroxypyruvate in the crystal structure of
2+
2+
octahedral Mn
complex with mixed oxygen Mg -SwHKA. Most importantly, addition of
Association of potentially bidentate phosphate to the holoenzyme both in the presence and
aldehyde acceptors (glycolaldehyde, absence of substrate did not alter the EPR spectra
ligands.[
26]
binding
methylglyoxal) did not significantly alter the EPR (figure 4, bottom), which confirmed our previous
spectrum of the holoenzyme (figure S33), while the notion that there is no direct interaction between the
donor substrates pyruvate and hydroxypyruvate metal and phosphate in the active site. A similar
considerably changed the EPR signal (figure 4, top). change in the zero-field splitting has previously been
The ketoacid functional group is therefore required reported for the Mn(II) bound extradiol cleaving
for coordination and activation of the donor substrate. catechol dioxygenase upon the addition of the
Pyruvate dramatically altered the spectrum, as the substrate 3,4-dihydroxyphenylacetate and for Mn(II)
higher order spin transitions with Δm
dominant. The EPR spectrum of pyruvate bound or phosphoenolpyruvate.
Mn-SwHKA exhibits features at g = 1.13, 1.44 2.16, pyruvate bound Mn-SwHKA have also been reported
.81 and 4.3 that represent higher order spin for oxalate decarboxylase, oxalate oxidase,
s
= 1 became bound pyruvate kinase upon the addition of pyruvate
[27]
Spectra similar to the
2
transitions. The feature around g = 4.3 exhibits the hydroxynitrile
characteristic 6-line pattern with a hyperfine coupling phosphoglucosemutase.
constant of circa 95 Gauss. This is indicative of a
lyase
GtHNL
and
[20, 28]
2
+
large change in the electronic structure of the Mn
Kinetic analysis of phosphate activation
On a mechanistic level, the aldol reaction between
(
hydroxy-)pyruvate and an aldehyde acceptor is fully
reversible via the same reaction pathway and
comprises of three distinct reaction steps, starting
from the bound substrate (Michaelis complex). These
steps are characterised by their corresponding rate
constants (k
initialised by deprotonation at C3 in the donor
substrate (k ) to afford an enolate intermediate.
Nucleophilic attack on the aldehyde then creates a
new C-C bond (k ) and final protonation of the
alkoxide closes the catalytic cycle (k
not shown).
1
– k
3
and k-1 – k-3). The reaction is
1
2
3
, for simplicity
Scheme 2. Discrete reaction steps during the aldol reaction
of (hydroxy-) pyruvate and an acceptor aldehyde. The
corresponding thermodynamic equilibria can also be
described by the associated rate constants in the forward
and reverse direction.
Figure 4. EPR spectroscopy of Mn(II) bound aldolase, In deuterated buffers, the enzyme catalyses the H-D
showing the effect of donor substrates on the spectrum of exchange in the donor substrate in the absence of
2+
Mn -SwHKA (top) and no effect by the addition of KPi aldehydes, which allows to specifically study the
(
bottom). EPR conditions: microwave frequency, 9.402 impact of phosphate on the kinetic rate constants of
GHz; microwave power, 0.2 mW; modulation frequency, (de-)protonation (k and k-1).
1
1
3
00 kHz; modulation amplitude, 10 Gauss; temperature,
7 K.
5
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Advanced Synthesis & Catalysis
10.1002/adsc.201900205
CH-π interactions
Since SwHKA was previously shown to be
particularly good at utilising HPA in aldol reactions,
its active site geometry was compared to a previously
published model of active site conservation among
the 19 class II pyruvate aldolases that were found to
[10]
be active towards HPA.
While highly similar,
Scheme 3. SwHKA catalysed H-D exchange in (hydroxy-) residue F210 in SwHKA occupies the position of a
pyruvate in deuterated buffers using the non-paramagnetic generally conserved leucine residue as the sole
Mg2 as metal cofactor. This allows to specifically study
+
apparent deviation from the model. A similarly
enolisation as the first step in the aldol reaction. Complete
positioned phenylalanine residue was previously
exchange was observed for all enzyme variants, indicating
reported for the class I KDPG aldolase, where the
no strict preference for the enantiotopic hydrogens when
authors described its function to cap the pyruvate
R=OH.
[29]
methyl group.
In carbohydrate utilising enzymes,
CH-π interactions between aromatic residues and
electropositive saccharide C-H bonds have been
extensively investigated and are considered one of the
The aldolase catalysed decarboxylation of
oxaloacetate then represents an extended model
system, which also takes the C-C bond
forming/breaking step (k
case, irreversible
decarboxylation, Keq = 1.36 × 10 at pH 7.5, table S2)
2
and k-2) into account. In this key determinants for carbohydrate recognition in
[17, 30]
C-C
bond
breaking proteins.
Briefly, electron rich aromatic systems
6
(
donate electron density into electropositive C-H
bonds, thereby establishing binding interactions that
compensate for the mismatch in polarity. In analogy
to carbohydrate utilising enzymes, we therefore
hypothesised that CH-π interactions between F210
and the C-H bonds at C3 in HPA are responsible for
its efficient conversion by SwHKA. To provide
evidence for our hypothesis, we created the variant
F210L as a representative of the conserved active site
effects the direct formation of the active enolate
species. Subsequent protonation of the enolate
intermediate (k-1) then affords the final product
pyruvate.
[10]
geometry of pyruvate aldolases
F210Y and F210W which possess higher electron
densities in their aromatic system than
phenylalanine. To our surprise, all variants were
and two variants
Scheme 4. SwHKA catalysed retro-aldol type
[17]
decarboxylation of oxaloacetate. C-C bond breaking (k-2
directly affords the enol intermediate and the subsequent
protonation step corresponds to rate constant k-1
)
comparably able to catalyse the formation of the enol
intermediate from hydroxypyruvate during H-D
exchange experiments (figure 5), where variant
.
Remarkably, the presence of phosphate increases the
initial rate of H-D exchange in pyruvate by 120-fold
(
figure S31). In contrast, the K for oxaloacetate was
M
unaffected by the presence of phosphate, while the
catalytic rate constant kcat only increased 10-fold.
Phosphate therefore does not improve the binding of
the substrate, but exclusively increases its rate of
decarboxylation. In summary, these findings indicate
that when oxaloacetate is used as substrate,
(
de-)protonation is rate-limiting in the absence of
phosphate, while C-C bond formation/breaking
becomes rate limiting in its presence. For acceptor
substrates, where electronic effects or steric
constraints do not already render C-C bond formation
rate limiting to begin with, a theoretical rate
enhancement of up to 120-fold could be achieved by
increasing the rate of enolisation when phosphate
saturated conditions are used.
Figure 5. Enzyme catalysed H-D exchange in
hydroxypyruvate by SwHKA F210X mutants in deuterated
buffer (5 mM KPi, pD = 7.4). Control experiments in the
absence of enzyme showed <1% background exchange
over the investigated time frame.
6
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Advanced Synthesis & Catalysis
10.1002/adsc.201900205
F210L showed 50% and F210Y 175% relative Upon deprotonation, the enol intermediate then
activity with respect to the WT. The F210W mutant adopts a planar configuration and docking studies
was only slightly better than F210L and showed 60% suggest a newly formed hydrogen bond between the
relative activity, which could either be due to its steric hydroxyl group and R69, alongside CH-π interactions
constraints, or binding interactions which are too of the olefinic hydrogen with F210 (figure 6). This
strong for efficient turnover. Nevertheless, substantial geometry allows for the acceptor substrate to freely
differences were observed when the previously converge towards the nucleophile and facilitates C-C
reported conversion of hydroxypyruvate with bond formation. In the F210Y mutant, the phenolic
DL-glyceraldehyde[
10]
was followed over time. hydroxyl group is too far away for direct interactions
Equilibrium conditions were reached within 30 with the substrate and the observed increase in H-D
minutes and 1.5 hours for the F210Y mutant and the exchange rate must therefore originate from enhanced
WT respectively, whereas the F210L mutant took 72 CH-π interactions. Based on the configuration of
hours (figure S37). To rule out any possible effects L216 in the DDG aldolase (1dxf.pdb, rmsd = 1.61 Å),
from the acceptor substrate, the conversion of DL- the F210L mutation was modelled in silico as the
glyceraldehyde with pyruvate was followed under corresponding rotamer. The model revealed steric
identical conditions. In this case, no apparent clashes between the isobutyl side chain and the
difference was observed between F210L and the WT, hydroxyl group, which would prevent both
demonstrating that the observed effect indeed hydroxypyruvate and the enol intermediate from
originated from the different donor substrates (figure adopting similar configurations (figure S42). While
2+
S36 – S39). Soaking of Mg -SwHKA crystals with the F210L mutant can catalyse the first step in the
hydroxypyruvate showed that both C-H bonds at C3 aldol reaction, it subsequently seems to trap the enol
indeed are oriented towards the aromatic system of intermediate in a disfavoured configuration that
F210 for CH-π interactions and revealed a hydrogen hinders C-C bond formation.
bond between the substrate’s hydroxyl group and the
backbone carbonyl of I167 (figure 1). In this
Conclusion
configuration, the hydroxyl group is oriented towards
the inside of the active site, preventing it from
blocking the narrow substrate channel.
In summary, this characterization of the SwHKA
aldolase A5VH82 has yielded new insights into the
enzyme’s kinetic properties and the mode of
phosphate binding and activation. Due to the lack of
proximal amino acid residues, a structural molecule of
water was previously identified to be responsible for
deprotonation of the substrate. However, this water
molecule is only insufficiently activated by the
adjacent residues and renders deprotonation of the
donor substrate rate limiting. Alternatively, this
enzyme can bind inorganic phosphate via H44 with
high affinity, which replaces water as the catalytic
base and results in a rate enhancement of up to
1
20-fold. Since C-C bond formation becomes rate
limiting under phosphate saturated conditions, the
overall obtained rate enhancement then depends on
the kinetic properties of the acceptor substrate. Our
results highlight a common pitfall that is responsible
for the previous misconception of the effect of
phosphate in class II pyruvate aldolases: excessive
phosphate concentrations negatively affect the
reaction rate by removing the metal cofactor. We
demonstrated that phosphate activation is not a unique
property of the αββα sandwich fold, but also is
functionally conserved within the same order of
magnitude in the distinct (α/β) barrel fold of the
8
Figure 6. Docking of the planar enol intermediate from
SwHKA aldolase. Future research into class II
pyruvate aldolases should therefore take the
possibility of phosphate activation into account.
While the conserved variant F210L efficiently
catalyses the first step of the aldol reaction to generate
the enol intermediate from HPA, its configuration
hinders subsequent C-C bond formation. So far, CH-π
2
+
HPA into the active site of Mg -SwHKA (6r62.pdb) with
YASARA. CH-π interactions are established between F210
and the olefinic C-H bond at C3 in HPA and the hydroxyl
group forms a H-bond to R69. For simplicity, the second
monomer has been omitted. Figures were created with
PyMol.[
16]
7
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Advanced Synthesis & Catalysis
10.1002/adsc.201900205
H-D exchange
interactions have mainly been investigated for
carbohydrate active enzymes. Our findings now
illustrate their importance for another enzyme class,
where they allow for the conversion of a non-natural
NMR spectra were recorded using an Agilent 400 MHz
1
(
H, 9.4 Tesla) spectrometer operating at 399.67 MHz for
1
H at 298 K and were subsequently interpreted using
6
substrate. By further increasing the electron density of MestReNova. A benzene-D capillary (Sigma-Aldrich) was
used for external locking and quantitation during water
suppression experiments with the PRESAT-PURGE pulse
sequence. Spectra were recorded using a recycle delay of 2
seconds and 32 repetitions. SwHKA (1.25 mg/mL) was
the aromatic system in the F210Y mutant, we
obtained a variant of 2-fold improved activity. CH-π
interactions should therefore also be included in
2
+
rational
enzyme
design
strategies
when incubated with Mg (2 mM) in potassium phosphate buffer
(
5 mM, pD = 7.4, prepared in D
2
O, lyophilised and re-
O). The reaction was initialised by the
electropositive C-H bonds are involved.
dissolved in D
2
addition of ketoacid substrate (50 mM). Controls in the
absence of enzyme under identical conditions showed <1%
chemical background activity over the investigated time
frame.
Experimental Section
Detailed protocols for the enzyme expression, purification
and the corresponding gene sequences are provided next to
additional graphs in the electronic supporting information.
Aldol reactions of (hydroxy-) pyruvate with
DL-glyceraldehyde
2
+
SwHKA (0.5 mg/mL) was incubated with Mg (2 mM),
DL-glyceraldehyde (50 mM) and the reaction was
initialised by the addition of ketoacid substrate (50 mM) in
potassium phosphate buffer (5 mM, pH 7.0, 1.5 mL, 25°C,
Chemicals were generally bought in the highest purity
commercially available and were used without further
purification, unless stated otherwise.
5
00 rpm). Samples were quenched by 1:1 dilution with
General oxaloacetate decarboxylase activity assay
trifluoroacetic acid (TFA, 0.2% v/v in H
enzyme was precipitated by centrifugation. Concentrations
SwHKA aldolase was incubated with NADH (0.5 mM), were determined by RP-HPLC (ICSep ICE Coregel-87H3
2
O) and the
2+
M
(1 mM), L-Lactic dehydrogenase type II (LDH, from column, 0.4x25 cm, Transgenomic, 0.1% v/v TFA, 0.8
rabbit muscle, 10 U/mL, as ammonium sulphate mL/min, 60°C, 210 nm) on a Shimadzu LC-20AD system
suspension, Sigma Aldrich) in triethanolamine buffer using external standards.
(
TEOA, 5 mM, pH = 7.5) and the reaction was initiated by
the addition of oxaloacetate (0.5 mM) to give a final
volume of 1 mL. The change in absorbance was followed
at 340 nm in PMMA cuvettes (800 rpm, 25°C) in triplicate,
Crystallisation and data collection
Well diffracting crystals of apo-SwHKA protein were
obtained at 277 K using the vapour diffusion method, from
drops resulting from mixing equal volumes of a protein
sample at 9.2 mg/ml with a crystallization solution
consisting of 0.44 M to 0.65 M of sodium citrate and 0.1 M
using
a
Cary 60 UV-Vis spectrometer (Agilent
Technologies) equipped with a TC 1 stirring unit (Quantum
Northwest). Curve fitting was performed with IGOR,
assuming Michaelis-Menten type kinetics.
HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic
Measurement of inorganic phosphate
acid) at pH 7.05. Crystals took 7days to appear and grew to
their final size within a few days. Crystals of the holo-
Ammonium molybdate (1.5 g, 12% w/v) was dissolved in SwHKA in complex with hydroxypyruvate were obtained
6
M sulfuric acid (125. mL). The assay solution was freshly by transferring crystals of apo-SwHKA into a new drop
prepared from sodium ascorbate (0.2 g in 10 mL H O), containing 0.7 M of potassium bromide (KBr), 0.1 M
sodium dodecylsulfate (1.25 mL, 10% in H O) and HEPES pH 7.05, 20 mM of magnesium chloride (MgCl
2
)
2
2
ammonium molybdate solution (1.25 mL). 400 µL sample and 150 mM of hydroxypyruvate. The crystals were left in
were mixed with 600 µL assay solution, incubated for 10 this new condition for a few hours and then were cryo-
minutes at room temperature and the absorbance was cooled using as cryoprotectant 20% of glycerol. X-ray
measured at 750 nm.
diffraction data was collected at beamline P13 operated by
EMBL Hamburg at the PETRA III storage ring (DESY,
[
32]
Hamburg, Germany).
The protein crystallized in the
EPR
cubic space group P 42 3 2, with unit-cell parameters of
approx. a = 116.6 Å, with one molecule in the asymmetric
unit and a solvent content of 47.5%.
EPR measurements were carried out using a Bruker
EMXplus 9.5 spectrometer and the following conditions:
9
.402 GHz microwave frequency, 0.2 mW microwave
Structure solution and refinement
power, 100 kHz modulation frequency, 10 Gauss
modulation amplitude at a temperature of 37 K. The
[
33]
microwave power was optimized by recording a 2D Data were indexed and integrated with XDS, scaled with
[
34]
powerplot from 0 to 40 dB using the Xenon software AIMLESS,
and the space group was determined with
[
35]
[36]
(
Bruker), which was analysed using a labview EPR POINTLESS
from CCP4 programs suite.
Data-
analysis program written by W.R. Hagen (to be published collection statistics are given in table S1. The structure was
elsewhere). The low temperature was maintained by solved by the molecular-replacement method with the
[
37]
boiling liquid helium and the cold helium vapour was program MOLREP
using the crystal structure of apo-
passed through a double wall quartz glass tube which was SwHKA as a search model (unpublished data). Model
[
31]
mounted and fitted in the rectangular cavity.
Samples building and refinement of ligand bound holo-SwHKA was
[38] [39]
were prepared in TEOA buffer (5 mM, pH = 7.5) to contain performed with COOT
and PHENIX,
respectively.
1
.2 mM of purified apo-enzyme (calculated for the The program PHENIX was used to refine atomic
2+
molecular weight of the monomer of 27.4 kDa), Mn (1 coordinates together with individual isotropic atomic
mM, 0.85 eq.), KPi (2 mM, 1.7 eq.) ketoacids/aldehyde displacement parameters. TLS thermal anisotropic
substrate (10 mM, 8.3 eq.) to afford a final volume of 200 parameterization was also included in the final stages of
µL.
refinement, with each monomeric subunit divided into
three TLS groups, as suggested by TLS Motion
[38, 40]
Determination.
A randomly selected 5% of observed
reflections were kept aside during refinement for cross-
validation. The positions of the magnesium ion and of the
8
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Advanced Synthesis & Catalysis
10.1002/adsc.201900205
hydroxypyruvate became evident after the first cycles of
refinement, based on electron density difference maps, and
both entities were modelled and refined with full
occupancy. Solvent molecules were included in the model
after a few rounds of refinement as well as bromide and
potassium ions that were located at the surface of the
protein. Structure refinement statistics are listed in table S1.
[
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Substrate docking
A simulation cell of 9 Å was defined around R69 in the
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10.1002/adsc.201900205
FULL PAPER
CH-π Interactions Promote the Conversion of
Hydroxypyruvate in a Class II Pyruvate Aldolase
Adv. Synth. Catal. Year, Volume, Page – Page
Stefan R. Marsden, Luuk Mestrom, Isabel Bento*,
Peter-Leon Hagedoorn, Duncan G.G. McMillan
and Ulf Hanefeld*
a
Biokatalyse, Afdeling Biotechnologie, Technische
Universiteit Delft, van der Maasweg 9, 2629HZ
Delft, The Netherlands. U.hanefeld@tudelft.nl, +31
1
5 278 9304
b
EMBL Hamburg, Notkestrasse 85, 22607 Hamburg,
Germany. ibento@embl-hamburg.de
1
1
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