Structural insights into substrate specificity in variants of N-acetylneuraminic acid lyase produced by directed evolution

Article abstract of DOI:10.1016/j.jmb.2010.08.008

The substrate specificity of Escherichia coli N-acetylneuraminic acid lyase was previously switched from the natural condensation of pyruvate with N-acetylmannosamine, yielding N-acetylneuraminic acid, to the aldol condensation generating N-alkylcarboxamide analogues of N-acetylneuraminic acid. This was achieved by a single mutation of Glu192 to Asn. In order to analyze the structural changes involved and to more fully understand the basis of this switch in specificity, we have isolated all 20 variants of the enzyme at position 192 and determined the activities with a range of substrates. We have also determined five high-resolution crystal structures: the structures of wild-type E. coli N-acetylneuraminic acid lyase in the presence and in the absence of pyruvate, the structures of the E192N variant in the presence and in the absence of pyruvate, and the structure of the E192N variant in the presence of pyruvate and a competitive inhibitor (2R,3R)-2,3,4-trihydroxy-N,N-dipropylbutanamide. All structures were solved in space group P2₁ at resolutions ranging from 1.65 A to 2.2 A. A comparison of these structures, in combination with the specificity profiles of the variants, reveals subtle differences that explain the details of the specificity changes. This work demonstrates the subtleties of enzyme-substrate interactions and the importance of determining the structures of enzymes produced by directed evolution, where the specificity determinants may change from one substrate to another.

Full text of DOI:10.1016/j.jmb.2010.08.008

doi:10.1016/j.jmb.2010.08.008
J. Mol. Biol. (2010) 404, 56–69
Contents lists available at www.sciencedirect.com
Journal of Molecular Biology
journal homepage: http://ees.elsevier.com.jmb
Structural Insights into Substrate Specificity in
Variants of N-Acetylneuraminic Acid Lyase
Produced by Directed Evolution
Ivan Campeotto1, Amanda H. Bolt1,2, Thomas A. Harman1,2
,
Caitriona Dennis1, Chi H. Trinh1, Simon E. V. Phillips1, Adam Nelson1,2
,
Arwen R. Pearson1 and Alan Berry1
1Astbury Center for Structural Molecular Biology, Garstang Building, University of Leeds, Leeds LS2 9JT, UK
⁎
2School of Chemistry, University of Leeds, Leeds LS2 9JT, UK
Received 15 May 2010;
received in revised form
3 August 2010;
accepted 5 August 2010
Available online
6 September 2010
The substrate specificity of Escherichia coli N-acetylneuraminic acid lyase
was previously switched from the natural condensation of pyruvate with
N-acetylmannosamine, yielding N-acetylneuraminic acid, to the aldol
condensation generating N-alkylcarboxamide analogues of N-acetylneur-
aminic acid. This was achieved by a single mutation of Glu192 to Asn. In
order to analyze the structural changes involved and to more fully
understand the basis of this switch in specificity, we have isolated all 20
variants of the enzyme at position 192 and determined the activities with a
range of substrates. We have also determined five high-resolution crystal
structures: the structures of wild-type E. coli N-acetylneuraminic acid lyase
in the presence and in the absence of pyruvate, the structures of the E192N
variant in the presence and in the absence of pyruvate, and the structure of
the E192N variant in the presence of pyruvate and a competitive inhibitor
(2R,3R)-2,3,4-trihydroxy-N,N-dipropylbutanamide. All structures were
solved in space group P21 at resolutions ranging from 1.65 Å to 2.2 Å. A
comparison of these structures, in combination with the specificity profiles
of the variants, reveals subtle differences that explain the details of the
specificity changes. This work demonstrates the subtleties of enzyme–
substrate interactions and the importance of determining the structures of
enzymes produced by directed evolution, where the specificity determi-
nants may change from one substrate to another.
Edited by I. Wilson
Keywords:
directed evolution;
X-ray crystallography;
N-acetylneuraminic acid
lyase;
substrate specificity;
protein engineering
© 2010 Elsevier Ltd. All rights reserved.
*Corresponding author. E-mail address: a.berry@leeds.ac.uk.
Present addresses: I. Campeotto, Randall Division for Cellular and Molecular Biophysics and Cardiovascular Division,
New Hunt's House, King's College London, Guy's Campus, London SE1 1UL, UK; S. E. V. Phillips, Rutherford Appleton
Laboratory, Research Complex at Harwell, Harwell Science and Innovation Campus, Didcot, Oxon OX11 0FA, UK.
Abbreviations used: NAL, N-acetylneuraminic acid lyase; ManNAc, N-acetylmannosamine; Neu5Ac, N-
acetylneuraminic acid; DPAH, (5R,6R)-7-(dipropylamino)-4,5,6-trihydroxy-2,7-dioxoheptanoic acid; DHOB, (2R,3S)-2,3-
dihydroxy-4-oxo-N,N-dipropylbutanamide; THB, (2R,3R)-2,3,4-trihydroxy-N,N-dipropylbutanamide; PDB, Protein Data
Bank; PEG, polyethylene glycol.
0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.

Variants of N-Acetylneuraminic Acid Lyase
57
Introduction
Results and Discussion
N-Acetylneuraminic acid lyase (NAL; EC 4.1.3.3,
otherwise known as N-acetylneuraminic acid
aldolase or sialic acid aldolase) catalyzes the
reversible aldol condensation of N-acetylmannosa-
mine (ManNAc) with pyruvate to yield the most
abundant sialic acid N-acetylneuraminic acid
(Neu5Ac) (Fig. 1). Sialic acids are essential compo-
nents of complex carbohydrates that play pivotal
roles as recognition signals in a variety of biological
processes.2 Such molecular recognition events are
well exemplified in host–pathogen and host–
parasite interactions where the oligosaccharide is
often required for the invasion, infectivity, and
survival of the invading organism in the host (for a
review of the roles of sialic acids, see Rosenberg3).
Sialic acid mimetics can, therefore, be important
chemotherapeutic agents4,5 (e.g., the anti-influenza
drugs Relenza and Tamiflu).6 NAL has been used in
a number of syntheses of such analogues;7 however,
its synthetic utility has been limited by its substrate
specificity (e.g., two-carbon and three-carbon alde-
hyde acceptors are very poor substrates)8 and by the
poor facial stereoselectivity of wild-type NAL9 and
other members of the NAL superfamily during
carbon–carbon bond formation.10 To overcome
these problems, we have used structure-guided
protein engineering and directed evolution of the
Escherichia coli enzyme to create a pair of comple-
mentary biocatalysts to synthesize either possible
diastereoisomeric aldol product11 or to tailor the
enzyme for the parallel synthesis of sialic acid
mimetics.1,12 We have reported that the engineered
NAL variant E192N catalyzes the reversible cleav-
age of N-dialkylcarboxamide analogues of Neu5Ac
—such as (5R,6R)-7-(dipropylamino)-4,5,6-trihy-
droxy-2,7-dioxoheptanoic acid (DPAH) into pyru-
vate and (2R,3S)-2,3-dihydroxy-4-oxo-N,N-
dipropylbutanamide (DHOB) (Fig. 1)—five times
more effectively that the wild-type enzyme cata-
lyzes the cleavage of Neu5Ac.1 The residue
responsible for this alteration of specificity at
position 192 lies at one end of the active-site cleft
opposite the active-site catalytic residue Lys165,
which forms a Schiff base with the substrate as part
of the catalytic cycle (Fig. 2a). In order to probe the
mechanism by which alteration of the residue at
position 192 affects substrate specificity, we have
undertaken an analysis of the activity of all 20
variants of NAL at position 192 along with an X-ray
structural analysis of the E192N enzyme in the
absence and in the presence of a competitive
inhibitor, (2R,3R)-2,3,4-trihydroxy-N,N-dipropylbu-
tanamide (THB) (Fig. 1). These studies have
provided new insights into the binding of sub-
strates to the wild-type and variant enzymes and
suggest the structural basis for the discrimination of
substrates by the E192X variants.
The importance of various interactions between
wild-type NAL and different substrates or substrate
analogues has been revealed by an analysis of the
substrate specificity of wild-type NAL13 and by
X-ray crystal structures. In particular, the structures
of E. coli NAL in the absence of any substrate,14 the
hydroxypyruvate or sodium-borohydride-reduced
pyruvate complexes,15 and Haemophilus influenzae
NAL in the absence of substrate or in the presence of
sialic acid alditol (Neu5Ac2ol), 4-deoxysialic acid
(4d-Neu5Ac), or 4-oxo-sialic acid (4oxo-Neu5Ac)16
have revealed that the residues at positions 191, 192,
and 208 (E. coli numbering is used throughout this
article) interact strongly with the hydroxyl groups at
C7, C8, and C9 of Neu5Ac (Fig. 2). Saturation
mutagenesis of these residues in the E. coli
enzyme1,12 allowed us to identify residue 192 as
crucial in switching the substrate specificity from
Neu5Ac to DPAH.1
Activity profile of NAL variants at position 192
In order to determine the relationship between the
amino acid at position 192 and the activity with
various compounds, we sequenced 96 clones from
an E192X saturation mutagenesis library.1 This
allowed us to identify 15 of the 20 possible variants
of NAL at position 192. The remaining variants
(E192A, E192F, E192H, E192Q, and E192Y) were
then constructed by site-directed mutagenesis. All
variants of the E192X library were expressed in E.
coli as hexa-His-tagged proteins and purified to
homogeneity on Ni-NTA resin. The activity of each
enzyme for the cleavage of either Neu5Ac or DPAH
was determined at a single low concentration of
substrate (0.5 mM) so that the enzymes were
unlikely to be saturated (the Km of the wild-type
enzyme for Neu5Ac is ∼4 mM1), and the observed
rates may be an approximation of kcat/Km (Fig. 3).
These results show that the activity towards
Neu5Ac is highest for the wild-type enzyme
(E192), and only the E192Q variant shows signifi-
cant activity towards this substrate. The steady-state
kinetic parameters kcat and Km were then deter-
mined for the cleavage of Neu5Ac with several NAL
variants, as shown in Table 1. These results show
that correct interaction between Neu5Ac and resi-
due 192 is extremely important for binding and
catalysis. In the X-ray crystal structure of H.
influenzae NAL in complex with 4-oxo-Neu5Ac,16
residue Glu192 hydrogen bonds with the diol at C8
and C9 of the sialic acid analogue (Fig. 2), and it is
likely that the glutamine introduced in E192Q
mimics this interaction so well that the kinetic
parameters of the E192Q enzyme with Neu5Ac
(Table 1) are not significantly altered with respect to
wild type. This profile of activity, with only E192

58
Variants of N-Acetylneuraminic Acid Lyase
Fig. 1. Wild-type NAL catalyzes
the reaction of pyruvate (red) with
ManNAc. The specificity of this
reaction was altered1 to catalyze the
reaction of pyruvate with DHOB by
the mutation of Glu192 to Asn. Inset:
The structure of the competitive
inhibitor THB. Carbon atoms are
numbered for Neu5Ac, DPAH,
DHOB, and THB. Note that, upon
aldol condensation, C1, C2, C3, and
C4 of DHOB become C7, C6, C5, and
C4 of DPAH, respectively.
and E192Q producing significant activity, explains
the distribution of activities towards Neu5Ac found
during the initial isolation of a NAL variant capable
of turning over the dipropylamide DPAH.1 Inter-
estingly, maintaining the hydrogen-bonding capac-
ity at position 192 while reducing the length of the
side chain (e.g., in the E192D or E192N variants)
severely reduced the activity (kcat/Km) to less than
15% of the wild-type value with Neu5Ac (Table 1).
This is presumably because the substrate, in its
extended conformation, cannot span from the
introduced aspartate or asparagine to lysine 165,
where it has to form a Schiff base. This finding is also
in line with previous studies that have shown that
short carbon chain analogues of Neu5Ac are not
substrates for the wild-type enzyme.8
The activity profile of the E192X library with
DPAH differs dramatically from that with Neu5Ac,
as many more of the variants (e.g., E192F, E192H,
E192M, E192N, E192Q, E192P, and E192V) show
significant activity with the new substrate (Fig. 3).
The steady-state kinetic parameters of these
enzymes (Table 1) confirmed that the E192N
enzyme had the highest activity for DPAH. It is
interesting to note that E192Q shows a high level of
activity with DPAH, making this enzyme the most
promiscuous in terms of substrate specificity. All of
the active variants showed kcat values generally
similar to those of the wild-type enzyme, and
changes in the specificity constant kcat/Km were all
brought about by changes in Km for the substrate
(Table 1), implying alterations in the ability of the
enzyme to recognize and bind the substrate depend-
ing on the diversity of functionality at position 192.
The range of active functionalities at position 192
suggests that the interaction with the substrate in
these enzymes is less specific than the interaction
between E192 and Neu5Ac, and that a number of
residues are able to make interactions with DPAH to
promote catalysis. Interestingly, some of the active
variants have large hydrophobic side chains at
position 192 (E192F, E192Y, and E192M), and we
reasoned that these side chains may provide a
hydrophobic environment with which the propyl
groups of DPAH are able to interact in a favorable
manner. In order to gain further insights into the
mechanism of substrate specificity in the E192
variants, we embarked on an X-ray crystallographic
analysis of the ‘best’ enzyme for dipropylamide
cleavage (namely E192N) and on a structural
modeling of the possible binding modes of interac-
tion of DPAH with other E192 variants.
Crystallographic results
NAL has a (β/α)8 barrel structure of the type first
found in triose phosphate isomerase,17 and crystal
structures of E. coli NAL in the absence of ligands
[Protein Data Bank (PDB) code 1NAL],14 in complex
with hydroxypyruvate (PDB code 1FDY), or
trapped by sodium borohydride in the presence of
pyruvate (PDB code 1FDZ)15 were already available
in the PDB database. In addition, the structure of an
L142R mutant of E. coli NAL, which was evolved in
vitro to have increased dihydrodipicolinate synthase
activity, was also available in complex with hydro-
xypyruvate (PDB code 1HL2).18 We reasoned that a
structural understanding of the features that caused
the change in the specificity of the E192N variant
away from the reaction of pyruvate with ManNAc
towards that with DHOB would only be forthcom-
ing if we could solve the structure of NAL in the
presence of substrates or substrate analogues that
mimicked the full-length substrate. However, with
the exception of the L142R mutant NAL structure,18
E. coli NAL crystals had been obtained after crystal
growth in high-salt concentrations, resulting in the
presence of a sulfate ion in the catalytic pocket that

Variants of N-Acetylneuraminic Acid Lyase
59
Fig. 2. (a) Schematic showing
the interactions between residues
Asp191, Glu192, and Ser208, and
ManNAc. A Schiff base is formed
between pyruvate and Lys165. (b)
X-ray crystal structure of NAL from
H. influenzae in complex with the
substrate analogue 4-oxo-sialic acid
(PDB code 1F7B). The ketone at C4
is hydrated. The covalent bond
with residue Lys165 is visible, and
distances between the heteroatoms
in residues Glu192, Asp191, and
Ser208, and the heteroatoms in 4-
oxo-sialic acid are highlighted.
precluded co-crystallization with ligands larger than
pyruvate.14,16 We therefore identified new condi-
tions for the crystallization of E. coli NAL (in space
group P21) that avoided the use of sulfate ions and
allowed us to solve the structures of both the wild
type and the E192N variant in the presence and in
the absence of a number of ligands.
The crystal structures of wild-type E. coli NAL and
the E192N variant were solved at 2.2 Å and 1.8 Å,
respectively (Table 2), and show a tertiary structure
identical to that previously observed.14 The overall
RMSD between the Cα atoms in the wild-type
structure and the Cα atoms in the E192N structure is
0.3 Å. The only significant differences are the
orientation of Asn192 compared to that of the
wild-type Glu192 and the resulting changes in
surface potential at the entrance of the catalytic
pocket when the negatively charged glutamate is
replaced (Fig. 4).
Lys165, and the planarity of electron density is
consistent with the formation of the natural pyru-
vate enamine intermediate (Figs. 2a and 5). We have
previously reported the structure of the E192N
variant, in space group P212121, in complex with
pyruvate.19 The interactions made by pyruvate in
that structure and in the P21 structures of the wild-
type–pyruvate and E192N–pyruvate complexes
reported here are identical. The carboxylate group
of pyruvate forms hydrogen bonds with the
backbone amides of Ser47 and Thr48 and also with
the side-chain OH of Thr48 (Fig. 5), as previously
found for pyruvate and hydroxypyruvate15 and as
is typical of a number of related pyruvate-dependent
aldolases.16 Comparison with the apo-NAL struc-
tures indicates that there are no significant changes in
the active site upon pyruvate binding; the only
difference in the two structures appears to be a
further disordering of the C-terminal residues in a
remote location from the catalytic pocket.
The structures of complexes with pyruvate were
also obtained (to 1.65 Å resolution for the wild-type
enzyme and to 1.8 Å resolution for E192N; Table 2).
As expected, pyruvate was covalently linked to
Soaking the E192N variant with the substrate
DPAH in order to trap a DPAH–NAL complex was
unsuccessful due to the rapid rate of the cleavage

60
Variants of N-Acetylneuraminic Acid Lyase
Fig. 3. Activity of NAL variants
isolated and purified from the E192X
saturation mutagenesis library with
(a) Neu5Ac (0.5 mM) and (a) DPAH
(0.5 mM). Each assay was carried out
as described in Materials and Meth-
ods and contained ∼0.03 mg of
purified enzyme.
reaction. As a result of this, only density for the
cleavage product (pyruvate covalently linked to
Lys165) was observed, and this structure was
identical with the E192N–pyruvate complex de-
scribed above (data not shown). To overcome this
problem, we designed an analogue of the acceptor
DHOB (Fig. 1) in the E192N-catalyzed reaction. This
compound, THB (Fig. 1), mimics the substrate
DHOB, but the reactive aldehyde has been reduced
to an alcohol, preventing nucleophilic attack of
pyruvate enamine. THB was synthesized,20 and
steady-state enzyme kinetics showed that it is a
competitive inhibitor of the E192N NAL reaction
(Fig. 6). The Ki of THB for the reaction was measured
as 1.3 mM.
and, overall, appeared identical with the structure
of E192N in complex with pyruvate, with addi-
tional electron density only observed in the active
site. This strong electron density corresponds to the
pyruvate enamine with residue Lys165. The pla-
narity of this electron density confirms the
expected sp2 hybridization for the carbon–carbon
double bond of the enamine intermediate. The
noncovalent interactions of pyruvate with the pro-
tein are identical with those observed for the wild-
type–pyruvate and E192N–pyruvate complexes.
The Fo −Fc electron density map also revealed a
further strong positive electron density in the
enzyme active site. The inhibitor THB fits well
into this density, with the exception of the terminal
ends of the two propyl groups, likely due to the
freedom of rotation of these groups (Fig. 7). There
The structure of the E192N complex with
pyruvate and THB was solved at 2.05 Å resolution

Variants of N-Acetylneuraminic Acid Lyase
61
Table 1. Steady-state kinetic parameters of wild-type and E192X variants with Neu5Ac and DPAH
Neu5Ac
DPAH
kcat/Km
kcat/Km
(min−1 mM−1
)
Enzyme
kcat (min−1
)
Km (mM)
(min−1 mM−1
)
kcat (min−1
)
Km (mM)
Wild type
E192N
E192Q
E192V
E192F
E192H
E192M
E192P
260 6a
170 10a
300 20
120 8a
4.4 0.3a
59a
4.4a
85
3.5
27
19
14
4
73 4a
11 2a
7a
340a
250
270a
310
80
38 5a
130 3a
0.4 0.04a
0.7 0.05
0.4 0.1a
0.3 0.02
0.5 0.04
0.2 0.03
0.2 0.02
1.1 0.05
0.6 0.04
3.5 0.5
34 5a
5.9 0.4
6.2 0.7
170
95 4a
88
4
160
120
70
5
5
4
1
5
3
2
38 1.0
5
6
0.8
0.9
70
49
6
3
320
260
5
25
1
E192D
E192S
120
50
4.6 0.5
0.8
26
8
0.1
26 0.7
6
42
The steady-state kinetic parameters of the wild-type and variant enzymes for Neu5Ac and DPAH cleavage were measured with a
standard coupled enzyme assay. Kinetic parameters ( standard error of the fit) were determined by fitting the data to the Michaelis–
Menten equation.
a
Data taken from Williams et al.1
Table 2. Crystallographic data collection and refinement statistics
Wild type+
pyruvate
E192N+
pyuvate+THB
Wild type
E192N
E192N+pyruvate
Diamond beamline
Space group
a, b, c (Å),
I02
P21
I03
P21
I04
P21
I03
P21
I03
P21
54.8, 142.2, 84.2
109.0
79.56–2.20
(2.26–2.20)
0.102 (0.365)
0.063 (0.219)
8.5 (3.4)
54.7, 142.5, 83.6
109.2
54.6, 142.8, 84.5
109.0
47.60–1.80
(1.90–1.80)
0.086 (0.388)
0.054 (0.033)
8.3 (2.8)
56.9, 143.0, 83.9
109.8
79.06–1.80
(1.95–1.80)
0.070 (0.430)
0.043 (0.269)
12.0 (2.5)
57.0, 143.7, 83.3
109.9
79.31–2.05
(2.16–2.05)
0.09 (0.438)
0.056 (0.273)
8.6 (2.6)
β (°)
Resolution (Å)
79.1–1.65
(1.74–1.65)
0.060 (0.317)
0.038 (0.215)
13.5 (3.2)
Rmerge
Rpim (all I+ and I−)
〈I〉/σ〈I〉
Completeness (%)
Redundancy
99.9 (100)
3.7 (3.7)
91.2 (60.8)
3.4 (2.9)
98. 4 (97.2)
3.6 (3.5)
100 (99.9)
3.6 (3.5)
98.6 (96.3)
3.5 (3.4)
Wilson B-factor (Å2)
Number of reflections
Number of unique
reflections
34.8
31.7
26.8
21.8
24.5
197,621 (29,177)
54,006 (7847)
449,770 (37,158)
131,694 (12,746)
398,910 (55,837)
110,985 (15,919)
391,185 (54,278)
107,356 (15,671)
277,866 (37,606)
78,597 (11,141)
Resolution (Å)
79.56–2.20
(2.26–2.20)
0.211 (0.259)
0.271 (0.356)
0.338
79.1–1.65
(1.69–1.65)
0.209 (0.261)
0.247 (0.344)
0.334
47.60–1.80
(1.90–1.80)
0.197 (0.261)
0.253 (0.391)
0.463
79.06–1.80
(1.95–1.80)
0.188 (0.301)
0.225 (0.344)
None
79.31–2.05
(2.16–2.05)
0.190 (0.281)
0.234 (0.337)
0.06
R-factor
Rfree
Twinning fraction
Twinning operator
Number of atoms
Protein
−h, −k, h+l
−h, −k, h+l
−h, −k, h+l
None
−h, −k, h+l
9166
169
—
9166
758
9178
316
4 (Cl)
9292
654
9246
540
60 (THB)
Water
Ligands
31 (PEG 400, Na+)
24 (PEG 400,
pyruvatea)
Average B-factors (Ǻ2)
Protein
Waters
Ligands
RMSDs
32.9
27.9
—
22.8
26.7
32.4
28.0
26.7
26.3
22.8
27.2
45.8
28.4
31.9
50.1
Bond lengths (Å)
Bond angles (°)
Ramachandran plot, most
favored region (%)
PDB code
0.013
1.46
0.012
1.80
0.011
1.33
0.009
1.14
0.012
1.28
94.2b
98.3b
97.6b
98.0b
98.3b
2WO5
2WNN
2WNQ
2WNZ
2WPB
a
Two additional molecules of noncovalently bound pyruvate are observed in subunits C and D.
In all structures, 100% of residues lie within favored regions of the Ramachandran plot.
b

62
Variants of N-Acetylneuraminic Acid Lyase
Fig. 4. Close-up view of the active-site cleft of wild-type NAL (a) and the E192N variant (b). The backbone is shown as
a gray cartoon, and active-site residues are shown as sticks colored by atom type. (c) Wild type and (d) E192N are the
same view and show the solvent-accessible surface colored by electrostatic potential (calculated in PyMOL; blue, positive;
red, negative) to illustrate the change in surface potential as a result of glutamate-to-asparagine mutation, as well as the
position of residue 192 at the entrance to the active-site cleft.
was some variation in the quality of the electron
density and in the final modeled orientation for
THB in the four crystallographically independent
subunits of the tetramer, with subunit D showing
the most well-defined THB density. The electron
density in subunit B was more consistent with a
slightly different binding mode for THB in com-
parison to subunits A, C, and D (Fig. 8) (see the
text below and Table S1).
In all four subunits, H-bonds are formed between
the C4 hydroxyl group of THB and the backbone
amide of Ser208 and the side chain of Asp191 (Fig. 8).
In addition, the backbone carbonyl group of Gly189
makes H-bonds with the THB [with the C3 hydroxyl
group in subunits A, C, and D, and with the C2
hydroxyl in subunit B (equivalent to C5 and C6 of
DPAH)]. These interactions result in the more polar
portion of the inhibitor (containing the three hydroxyl
groups) being bound at the base of the catalytic
pocket next to the pyruvate (Figs. 7 and 8). During
enzyme catalysis, a new carbon–carbon bond is
formed between C3 from the pyruvate enamine and
the C4 from the aldehyde acceptor. In the enzyme–
pyruvate–THB complex structure, this bond cannot
be formed, as THB is unable to undergo nucleophilic
attack by the enamine C3. Nonetheless, the C3 of the
pyruvate enamine lies only ∼3.5 Å from the C4 of
THB, compatible with these two atoms being in van
der Waals contact. This distance compares well with
the separation of the substrate aldol donor and
acceptor molecules (3.3–4.4 Å) found in the X-ray
structure of a different aldolase, the Mycobacterium

Variants of N-Acetylneuraminic Acid Lyase
63
Fig. 5. Active site of the wild-
type NAL–pyruvate complex. Two
views of the active site of the wild-
type NAL–pyruvate complex
showing the interactions of the
pyruvate enamine with the en-
zyme. Pyruvate is covalently linked
to Lys165 and makes hydrogen
bonds with Ser47 and Thr48. The
hydrogen-bonding network to
Tyr137 and a conserved water
molecule are also shown. The
lower panel shows a rotation of
the molecule to display the planar
nature of the 2Fo −Fc electron den-
sity (contoured at 1 RMSD) of
pyruvate–enzyme bonding con-
firming the enamine structure for
this intermediate.
tuberculosis fructose bisphosphate aldolase,21 and
therefore represents the Michaelis entry (or exit)
complex in which the two substrates are ideally
placed, ready for the nucleophilic attack of the
enamine on the aldehyde of the acceptor substrate.
The structure of E192N, in complex with pyruvate
and THB, reveals the structural basis of the discrim-
ination between substrates manifested by wild-type
and E192N NAL. In the wild-type enzyme, residue
Glu192 is sandwiched between Tyr190 of the same

64
Variants of N-Acetylneuraminic Acid Lyase
Fig. 6. Lineweaver–Burk plot of
the steady-state enzyme kinetics of
E192N reaction in the absence and
in the presence of the inhibitor THB:
(●) absence of THB; (○) 0.25 mM
THB; (▼) 0.5 mM THB; (▽) 1 mM
THB; (■) 1.5 mM THB; (□) 2 mM
THB. The initial rates of reaction
were measured using the standard
enzyme assay, and kinetic para-
meters were determined by fitting
all data to the rate equation for a
single-substrate reaction in the
presence of a competitive inhibitor
to measure the Ki for THB.
subunit and Tyr172 of a neighboring subunit. In this
position, its carboxylic acid group points towards the
active-site cleft, perfectly positioned to form strong
hydrogen bonds with the O8 and O9 of the glycerol
moiety of the incoming substrate Neu5Ac (or the
equivalent O5 and O6 of ManNAc). In contrast, in the
E192N variant, the amide of Asn192 points out of the
active-site cleft (a position disfavored for glutamate
due to unfavorable contacts with the neighboring
hydrophobic residues Tyr172 and Ile243), and this
conformation produces a larger hydrophobic binding
surface that is ideal for interaction with THB. The two
conformations of THB (found in subunits A, C, and D
compared with subunit B; Fig. 8) occur because of a
rotation of the main-carbon chain of THB around the
C4–C3 bond. This places the two propyl arms in
different positions in the two conformations. In
subunits A, C, and D, the syn arm of the dipropyla-
mide lies against the hydrophobic surface of Tyr190,
Ile247, and Tyr172 (this latter residue is from a
neighboring subunit), while the anti arm lies in a deep
hydrophobic pocket composed of Ile243, Thr209,
Val251, and Leu246. In this binding conformation, the
mutated residue at position 192 lies at the bifurcation
of the two propyl arms of THB. Modeling of a
glutamate residue at position 192 shows that the
surface of the wild-type enzyme would be more polar
in this important binding region and that there would
also be steric clashes between the enzyme carboxylate
and the C1 and C2 atoms of the dipropylamide arms.
In subunit B, the other conformation of bound THB
places the syn arm of the inhibitor in the same region
as in subunits A, C, and D, on the surface of Tyr190
and Ile247; however, in this case, the hydrophobic
pocket is empty, with the anti arm instead lying
against another hydrophobic part of the surface
formed from residues Val251 and Phe252. In this
case, the introduced Asn192 lies between the syn-
propyl arm and the amide carbonyl group. The
modeled position of the wild-type glutamate residue
again shows steric clashes of the Glu192 carboxylate,
this time with both the syn-propyl and the amide O
atom. With the assumption that the position and
orientations of the bound competitive inhibitor THB
accurately reflect the binding of the substrate DPAH,
the structural basis of discrimination against DPAH
binding to the wild-type enzyme and in favor of
binding to the E192N variant becomes clear. The
larger glutamate at position 192 in the wild-type
enzyme prevents DPAH binding not only through
steric clashes with the substrate, which cannot be
relieved because of the planarity of the substrate, but
also through the negative charge of Glu192 making
unfavorable interactions with the hydrophobic parts
of DPAH. An asparagine at position 192, on the other
hand, provides an uncharged surface with no steric
interference with DPAH binding. These results also
explain the substrate activity profile found previously
for the E192N variant.12 Dialkyl substituents, includ-
ing dibutylamides, are well accepted by the E192N
enzyme, and we would expect that the syn arm of
each amide would bind similarly to that of THB,
while the hydrophobic pocket formed from Ile243,
Thr209, Val251, and Leu246 described above is deep
enough to accommodate the longer butyl group. We
suggest that other active substrates, including pyrro-
lidinyl amide, morpholinyl amide, and piperidinyl
amide,1,12 interact with the E192N variant through
the extensive hydrophobic surface generated by
removing the charge from position 192, and this
explains why the more hydrophobic cyclic substrates
are preferred.
Understanding the role of residue 192 in
substrate selectivity
Catalysis of the aldol condensation/cleavage of
the natural substrates of NAL is supported by a

Variants of N-Acetylneuraminic Acid Lyase
65
Fig. 7. X-ray crystal structure of the E192N NAL–pyruvate–THB complex. (a) The active site of the E192N NAL
enzyme in the presence of pyruvate and the competitive inhibitor THB. The green net shows a THB electron density omit
map contoured at an RMSD of 1.5. Interactions of pyruvate enamine with Ser47 and Thr48, as well as the interactions of
THB with Ser208 and Asp191, are also shown. (b) Details of Lys165–pyruvate enamine and THB. The green net shows the
THB omit map as described above. 2Fo −Fc electron density contoured at an RMSD of 1.0 is shown as a blue net for the
Lys165–pyruvate Schiff base. The distance (3.6 Å) between the pyruvate methyl group and the C4 of THB (equivalent to
the new carbon–carbon bond formed between the substrate acceptor and the donor) is shown as a red dotted line.
narrow range of amino acids at position 192 (E and
Q; Fig. 3a) characterized by their polarity and size,
as described above. In contrast, the reaction with
DPAH and other dialkyl derivatives tolerates a
much wider range of side-chain size and polarity at
this position (Fig. 3b), with E192Q showing high
levels of activity with both natural and synthetic
substrate classes. Our structural investigations of the
E192N enzyme and, in particular, its complex with
the inhibitor THB have enabled us to propose a
general structural basis for these findings. The
successful binding and catalysis of the reaction
with dialkylamides clearly require a suitably sized
hydrophobic binding site. Very large residues at
position 192 (e.g., E192W) or charged residues
(E192R, E192K, E192, and E192D) would interfere
with substrate binding by breaking up the hydro-
phobic binding surface required, and this is reflected
in their low activity (Fig. 3b). On the other hand, we
suggest that small residues at position 192 (E192G,
E192A, and E192S) do not provide enough surface
for interaction with the substrate, explaining their
lack of activity. Of more interest is how larger
residues at position 192 (e.g., E192F, E192Y, E192Q,
E192M, E192V, and E192P) are still able to promote
reasonable levels of activity when we might have
expected these large residues to clash with the alkyl
arms of the substrate. However, in silico mutagenesis
of residue 192 revealed a number of side-chain
conformations that allow packing of the mutated

66
Variants of N-Acetylneuraminic Acid Lyase
Fig. 8. Schematic representation
of THB binding to E192N NAL.
Top: Binding of THB in subunits A,
C, and D. THB (black lines) binds in
the active-site pocket, with its C4
positioned 3.6 Å from the C3 of
pyruvate (red) bound as a Schiff
base to Lys165. Ser208 and Asp191
make H-bonds with the C4–OH
and C3–OH groups. Asn192 (or-
ange shaded box) lies between the
syn-propyl arm and the anti-propyl
arm. The hydrophobic sites (green
shaded boxes) for these arms are
formed from Tyr190, Ile247, and
Tyr172′ (syn), and a deep pocket is
formed by Leu246, Ile243, Thr209,
and Val251 (anti). In this binding
mode, Phe252 (gray shaded box)
appears to have little binding role.
Bottom: Binding of THB in subunit
B. Rotation of THB around the C3–
C4 bond compared with binding in
subunits A, C, and D positions THB
such that Ser208 and Asp191 now
H-bond to the C4–OH and C2–OH
groups and Asn192 lies between the
syn arm and the C1 carbonyl group.
This rotation positions the syn arm
in the same hydrophobic binding
site (green boxes); however, in this
orientation, the anti arm is bound
against Val251 and Phe252 (green
boxes), and the deep hydrophobic
pocket (gray boxes) is empty. Note that Val251 lies in a position where it can form part of the deep hydrophobic pocket
and the site for the anti arm in subunit B.
side chain into the overall protein structure without
significant clashes with the position of the THB
bound in our crystal structure. For example, both
Tyr192 and Phe192 can pack in a hydrophobic site
between residues Tyr172 and Tyr190, providing a
suitable hydrophobic surface for THB binding
without steric clashes. Similarly, Met192 can lie in
this region with the terminal methyl group rotated
away from the substrate to provide room for THB
binding, while smaller hydrophobic residues such
as valine and proline can also fit into the folded
chain at position 192 without serious distortions to
the backbone of the protein and without interfering
with the binding of THB and hence of the
dialkylamide substrates. A similar analysis reveals
the basis of the promiscuity of the E192Q enzyme
(Fig. 3a and b). If modeled in the same conformation
as the native glutamate, the introduced glutamine is
able to form H-bonds with the O8 and O9 hydroxyl
groups of the natural substrate Neu5Ac. However,
this conformation of the glutamine would cause
rejection of DPAH as a substrate because of steric
interference. On the other hand, an alternative
conformation of the neutral glutamine side chain,
in which it can pack between the two active-site
tyrosine residues in a conformation disfavored for
the negatively charged glutamate residue, is possi-
ble. In this conformation, it provides a hydrophobic
surface with no steric problems for DPAH binding.
In this way, the E192Q enzyme is able to provide
alternative modes of substrate binding that account
for its promiscuity.
Conclusions
The directed evolution approach for the con-
struction of new substrate specificities does not
require any previous structural or mechanistic
knowledge of catalysis in the target enzyme, and
sequence analysis of the resultant successful
variants selected often contains unexpected resi-
dues at unexpected positions to produce the
desired activity. However, a full understanding
of the mechanisms underlying the changes in
specificity is required if we are to learn how to
rationally produce new activities, and reports of
the structures of enzymes with altered substrate

Variants of N-Acetylneuraminic Acid Lyase
67
NaCl supplemented with protease cocktail inhibitor
(Roche). NAL was bound to Qiagen Ni-NTA resin
equilibrated in the same buffer containing 30 mM imid-
azole and was eluted at 500 mM imidazole. Purified NAL
was dialyzed overnight against 50 mM Tris–HCl and
150 mM NaCl (pH 7.5). Further purification was achieved
by size-exclusion chromatography using an S-200 gel-
filtration column (Amersham) in the same buffer before
storage and concentration. Protein purity was assessed by
SDS-PAGE, and molecular weight was confirmed with
electrospray ionization mass spectrometry.
specificity created by directed evolution are
appearing. These have revealed that altered
substrate specificity can be brought about in a
number of ways. For example, in aspartate
aminotransferase, changes in residues involved in
direct binding to the substrate22 or conformational
changes in the enzyme brought about by muta-
tions often at a distance of N10 Å from the active
site23 have been demonstrated to affect specificity.
Similarly, the promotion of a more compact confor-
mation of the acyltransferase LovD brought about by
mutations was found to be important in creating a
simvastatin synthase from this template.24 Finally,
significant changes in the size and conformation of
active-site residues resulting in the reshaping of the
active site were considered a major determinant of
the specificity switches found in evolved enantio-
selective epoxide hydrolase.25 Here, by combining
substrate specificity profiles with X-ray crystal
structure determination, we have been able to dissect
the way that the residue present at position 192 of
NAL can affect substrate specificity. The change
from Glu192 to Asn192 to switch the specificity from
Neu5Ac to DPAH was not a change we would have
rationally chosen to move from a triol-containing
substrate to the more hydrophobic and bulkier
dipropylamide. Our results have shown that when
the mutations at position 192 provide an extensive
hydrophobic surface capable of binding dipropyl-
containing substrates, there are a number of con-
formations of the substrate that result in activity.
However, if there is too much space between the
enzyme and the substrate (e.g., with small or
hydrophilic residues at position 192), then the H-
bonding at the other end of the substrate is not
sufficient to provide enough binding energy to hold
the substrate for catalysis to occur. This accords with
the lack of enzyme activity for ‘short’ two-carbon or
three-carbon aldehyde substrates.8 These results
point the way towards engineering NAL variants
with activity for such substrates, perhaps by reduc-
ing the size of the active site around the aldehyde
substrate binding pocket, and highlight the need for
future structural studies of evolved enzymes.
Enzyme assay
Aldolase activity with Neu5Ac and analogues was
measured using a coupled enzyme assay with lactate
dehydrogenase and NADH, as previously described.1,26
The assay was performed at 25 °C in a 1-mL cuvette
containing 50 mM Tris–HCl (pH 7.5), 0.2 mM NADH,
0.5 U of lactate dehydrogenase, and a suitable aliquot of
NAL (1–100 μg). The reaction was initiated by the addition
of varying concentrations of substrate (0.5–20 mM
Neu5Ac and 0.1–2.5 mM DPAH; chosen to bracket the
measured Km values). The decrease in absorbance at
340 nm was recorded on a Uvikon 930 spectrophotometer
as the measure of enzyme activity. One unit of aldolase
activity is defined as the amount of enzyme that catalyzes
the oxidation of 1 μmol min−1 NADH in this system, using
the molar extinction coefficient of NADH (6220 M−1 cm−1).
The triol, THB, was synthesized as previously described,20
and inhibition of the NAL reaction was measured by
including THB (0.25–2 mM) in the standard enzyme assay.
Steady-state kinetic parameters kcat, Km, and Ki were all
estimated by nonlinear regression to the appropriate rate
equations.
Construction of the E192X library
Preparation of the E192X saturation mutagenesis library
was undertaken using megaprimer PCR, as previously
described.1 DNA sequencing of 96 mutants revealed 15 of
20 different amino acids at position 192. The remaining
five mutants were constructed using QuikChange site-
directed mutagenesis, following the instructions provided
by the manufacturer (Stratagene).
Protein crystallization
Tests with the pre-crystallization assay kit (Hampton
Research) indicated an optimum NAL concentration of
10 mg mL−1. The initial conditions for crystallization were
identified with an Oryx crystallization robot (Douglas
Instruments) using commercial sparse matrix screens
(Hampton Research). Crystals were grown at 4 °C by
sitting drop diffusion at a ratio of 2 μl of protein (10 μg
mL−1) to 2 μl of mother liquor. Several hits were obtained,
but high-salt conditions (such as 3 M (NH4)2SO4 or 2 M
Li2SO4) were ignored on the basis of our previous studies
and the data reported in the literature.16 The final
optimized crystallization conditions were 100 mM Tris–
HCl (pH 8.0–8.30), 200 mM NaCl, and 18% (wt/vol)
polyethylene glycol (PEG) 3350, which yielded crystals in
7–10 days. This condition is more physiologically relevant
Materials and Methods
Expression and purification of NAL
E. coli BL21-competent cells were transformed with the
plasmid pKnanA harboring the wild-type NAL gene or
the E192X NAL library members, as previously
described.1 Cells were grown in 5 mL of 2YT medium
containing 50 μg mL−1 ampicillin and inoculated in 2 L of
2YT medium containing 50 μg mL−1 ampicillin and
0.4 mM IPTG for 16 h at 30 °C. Cells were lysed in a
Cell Disruption Systems apparatus at 19,000 psi in 50 mM
potassium phosphate buffer (pH 7.5) containing 300 mM

68
Variants of N-Acetylneuraminic Acid Lyase
than the published high-salt conditions16 and, crucially, it
allowed us to soak the substrate pyruvate and the triol
inhibitor in the crystals.
In all structures, except for that of the E192N variant in
complex with pyruvate, REFMAC detected a twinning
fraction between 6% and 46%, with a twinning operator
−h, −k, h+l. In these structures, refinement was performed
throughout using the twin refinement option in REFMAC5.
The crystals belonged to space group P21 with unit cell
dimensions a=57 Å, b=143 Å, c=83 Å, and β=109° (decimal
numbers are omitted due to their variability in several
structures). Occasionally, crystals also grew in space
group P212121, and we have recently reported the
structure of the E192N variant in complex with
pyruvate19 in this crystal form. To form the NAL and
the E192N variant pyruvate complexes, we soaked
crystals in mother liquor supplemented with 100 mM
sodium pyruvate (Sigma Aldrich) and 15% (vol/vol) PEG
400 (Hampton Research) for 1 min. The crystals were
then sequentially transferred into mother liquid contain-
ing 20% (vol/vol) PEG 400 (with 5% increments in PEG
400 concentration) and finally soaked in mother liquor
containing 25% (vol/vol) PEG 400 before being flash
cooled in liquid nitrogen. In the case of the pyruvate and
inhibitor complexes, the same sequential procedure was
performed with the addition of 70 mM THB inhibitor in
the last cryoprotection step [25% (vol/vol) PEG 400] for
2 min before flash cooling.
Accession numbers
Coordinates and structure factors have been deposited
in the PDB with accession numbers 2WO5 (wild type),
2WNN (wild type+pyruvate), 2WNQ (E192N), 2WNZ
(E192N+pyruvate), and 2WPB (E192N+pyruvate+THB).
Supplementary materials related to this article can be
found online a doi:10.1016/j.jmb.2010.08.008
Acknowledgements
We would like to thank Garib Murshudov for
discussions and for help with resolving the high
degree of twinning in some of our crystals. The
X-ray diffraction data described were collected with
the support of Diamond Light Source beamlines I02,
I03, and I04. A.R.P. was supported by a Research
Councils UK Academic Research Fellowship. We
also thank the Biotechnology and Biological Sciences
Research Council and The Wellcome Trust for
funding.
Data collection and structure solution
Diffraction data for each structure were collected from
single crystals on the macromolecular crystallography
beamlines of Diamond Light Source (stations I02, I03, and
I04) at 100 K. The data were indexed, integrated, and
scaled using MOSFLM27 and SCALA,28 and further
processed to generate a complete list of structure factors
with Rfree flags using the CCP4 suite (Table 2).29 Five
percent of the data were randomly selected and excluded
from the refinement to constitute the Rfree set. We have
recently reported the crystal structure of the E192N
variant in complex with pyruvate in the space group
P212121 (PDB code 2WKJ)19 at a relatively high resolution
(1.45 Å). This structure was used as a molecular
replacement model to solve the structure of the NAL
complex with pyruvate (PDB code 2WNN) in space
group P21 using Phaser.30 For the other structures
presented here (all belonging to space group P21), the
initial phases were calculated by combining the initial
model obtained from this molecular replacement and the
relevant recorded amplitudes after five cycles of rigid-
body refinement in REFMAC531 (treating the entire
tetramer as a rigid body).
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