Structure-guided redesign of d-fructose-6-phosphate aldolase from E. coli: Remarkable activity and selectivity towards acceptor substrates by two-point mutation

Article abstract of DOI:10.1039/c1cc11069a

Structure-guided re-design of the acceptor binding site of d-fructose-6-phosphate aldolase from E. coli leads to the construction of FSA A129S/A165G double mutant with an activity between 5- to >900-fold higher than that of wild-type towards N-Cbz-aminoal

Full text of DOI:10.1039/c1cc11069a

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COMMUNICATION
Structure-guided redesign of D-fructose-6-phosphate aldolase from
E. coli: remarkable activity and selectivity towards acceptor
substrates by two-point mutationw
a
a
a
Mariana Gutierrez,a Teodor Parella,b Jesu
´
s Joglar, Jordi Bujons and Pere Clapes*
´
Received 23rd February 2011, Accepted 31st March 2011
DOI: 10.1039/c1cc11069a
Structure-guided re-design of the acceptor binding site of
D-fructose-6-phosphate aldolase from E. coli leads to the
construction of FSA A129S/A165G double mutant with an
activity between 5- to >900-fold higher than that of wild-type
towards N-Cbz-aminoaldehyde derivatives.
Class I D-fructose-6-phosphate aldolase from E. coli (FSA)
was recently reported to stereoselectively catalyze (i.e. >50 : 1
syn/anti, 3S,4R : 3S,4S) the reversible aldol addition of
dihydroxyacetone (DHA), hydroxyacetone (HA) and hydroxy-
butanone (HB) to a structural variety of acceptor aldehydes.1,2
Importantly, FSA can also accept glycolaldehyde (GO) as a
donor, offering the possibility to perform stereoselective cross-
aldol additions of GO in water.3 Moreover, a single mutation,
FSA A129S, improved the reactivity towards DHA by
B17-fold in terms of kcat/KM, expanding the scope of the
preparative competences of the wild-type.4 Both biocatalysts
offer a great advantage over DHAP-dependent aldolases
because the direct use of unphosphorylated nucleophiles
simplifies enormously the synthesis by avoiding the extra
efforts in handling the phosphate group, unless a phosphory-
lated product in the specific C1 position is desired.
Scheme 1 Aldol addition of DHA and HA to selected aldehydes
catalysed by FSA (wt = wild-type).
and 2,5-imino-1,2,5-trideoxy-D-mannitol (6-deoxy-DMDP),
respectively.5 Alternatively, FruA from rabbit muscle (RAMA),
a DHAP-dependent aldolase, rendered 10–46% yield of
phosphorylated (5S)-5 and (5R)-5, respectively.6 FSA wild-type
gave also a disappointing 28–40%z yield in the addition of
DHA to relatively simple aldehydes such as O-benzyloxy-
acetaldehyde (3) and N-Cbz-glycinal (2).2 Aldol addition of
DHA to 2 furnishes the aldol adduct precursor of 1,4-dideoxy-
1,4-imino-D-arabinitol (DAB),2 which is a potent a-glucosidase
inhibitor, and a promising potential therapeutic candidate as an
anti-hyperglycemic agent.7
Despite the reported synthetic abilities of FSA and the A129S
mutant, limitations arising from the acceptor substrate tolerance
have been identified. For instance, no aldol addition of DHA
or HA to a-substituted aminoaldehydes, such as (S)- and
(R)-N-Cbz-alaninal ((S)-1 and (R)-1, respectively, Scheme 1),
was observed. The respective aldol adducts, (5S)-5 and (5R)-5,
are key intermediates for the expedient synthesis of the
pyrrolidine type iminocyclitols, 2,5-imino-1,2,5-trideoxy-D-glucitol
a
´
Instituto de Quımica Avanzada de Catalun˜a-CSIC, Jordi Girona 18-26,
In this work, we endeavoured to re-design rationally the
acceptor-binding site of the FSA wild-type and the FSA A129S
mutant by structure-guided site directed mutagenesis. Our goal
was to modify the acceptor aldehyde binding pocket of both FSA
enzymes to improve their catalytic performance towards N-Cbz-
aminoaldehydes without altering their high stereoselectivity,2,4
thus enlarging the number of tolerated substrates and expanding
their general utility in asymmetric carbon–carbon bond formation.
The crystallographic structure of FSA and the minimized
modelled hemiaminal of D-fructose-6-phosphate (F6P) and
K85 revealed two putative residues that might be related with
08034 Barcelona, Spain. E-mail: pere.clapes@iqac.csic.es;
Fax: +34 93 2045904; Tel: +34 93 4006112
b
`
`
Servei de Ressonancia Magnetica Nuclear, Dept of Chemistry,
Universitat Autonoma de Barcelona, Bellaterra, Spain
`
w Electronic supplementary information (ESI) available: General proce-
dures, site-directed mutagenesis, electrospray ionization mass spectro-
metry of the FSA mutants, specific aldolase activity of the FSA wild-type
and the mutants, preliminary screening of the aldol addition reactions
catalysed by FSA wild-type and mutants, analytical methods, determina-
tion of initial velocities and steady state kinetic parameters, preparation
of aldol adducts (5S)-5, (5S)-6, (5R)-5, (5R)-6, and 7 and conversion into
the corresponding iminocyclitols and copies of their NMR spectra
including aldol adduct 11. See DOI: 10.1039/c1cc11069a
c
5762 Chem. Commun., 2011, 47, 5762–5764
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the acceptor substrate-binding site (see Fig. 1A). Residue R134
appears to be involved in accommodating phosphorylated
acceptors such as the D-glyceraldehyde-3-phosphate, by analogy
to that equivalent of transaldolase B from E. coli,8 and thus
may interfere with large hydrophobic non-phosphorylated
aldehydes such as the ones selected in this study. Therefore,
substitution of alanine for arginine was envisaged as a way to
favour the accommodation of bulky substrates. The second
residue was the A165, which is oriented towards the covalent
complex between DHA/F6P and the conserved Lys residue.
Substitution of glycine for alanine may allow more space and
flexibility to allocate the aldehydes, especially those C-a
substituted. Therefore, FSA R134A and FSA A165G mutants
were constructed and investigated as biocatalysts for the aldol
addition of DHA and HA to aldehydes 1–4 (Scheme 1).
The results (Table 1 and ESIw) revealed that the FSA
A165G provided good to excellent conversions to aldol
adducts for the selected acceptor aldehydes, at initial velocities
from 4 to 175-fold higher than those of FSA wild-type either
with DHA or HA donors. Furthermore, both enantiomers of
C-a substituted Cbz-alaninal, which were not substrates for
the FSA wild-type, were tolerated as acceptors.
The FSA A129S mutant gave also improved conversions in
the aldol addition of DHA albeit it showed similar acceptor
selectivity as compared with FSA wild-type.4 FSA R134A
gave comparable or lower conversions than that with FSA
wild-type for the selected aldehydes (see ESIw). Then, we
reasoned that the higher affinity of FSA A129S for DHA
combined with the wider aldehyde tolerance of FSA A165G
might benefit from a synergistic effect of these two mutations.
Indeed, the FSA A129S/A165G double mutant resulted to be
the best biocatalyst for the aldol addition of DHA and HA to
the selected aldehydes. Unprecedented excellent results were
obtained for the aldol addition reactions of DHA to aldehydes
(S)-1 and (R)-1, whereas no reaction was detected using FSA
wild-type, FSA R134A or FSA A129S. Models of the covalent
complexes of adducts (5S)-5 and (5R)-5 in the active centre of
the A129S/A165G double mutant showed, as expected,
that the A165G mutation generates the required space to
allocate the C-a methyl group without clashing with the
Fig. 1 (A) Modelled F6P-derived hemiaminal (green) in the active
centre of FSA wild-type. Residues considered for mutation are shown
in yellow. (B) Modelled hemiaminal complexes of (5R)-5 (cyan) and
(5S)-5 (orange) in the active centre of the FSA A129S/A165G mutant.
Residues mutated are shown in yellow. An essential water molecule is
shown as a red ball. The surface of the protein close to residue 165
shows the increased available space in the double mutant relative to
the wild-type protein, which allows allocation of the C-a methyl group
of 5 without steric clashes with the protein.
Table 1 FSA-catalyzed aldol addition of DHA or HA to aldehydes 1–4. Initial reaction rates (no) and percentage of aldol adduct formation
noa/mmol minꢀ1 mgꢀ1
Aldol adduct,b % (protein used/mg)
Acceptor
aldehyde Donor
FSA wt FSA A129S FSA A165G FSA A129S/A165G FSA wt FSA A129S FSA A165G FSA A129S/A165G
(S)-1
DHA ndc
ndc
0.10 ꢁ 0.03 0.6 ꢁ 0.1
3.8 ꢁ 0.2 13 ꢁ 2
ndc(1.0) ndc(1.0)
31(0.5) 21(0.5)
ndc(1.0) ndc(1.0)
51(0.5)
85(0.05)
73(0.5)
84(0.05)
100(0.1)
100(0.1)
81(0.9)
95(0.05)
91(0.3)
90(0.1)
78(0.5)
92(0.05)
95(0.5)
95(0.05)
100(0.05)
100(0.01)
100(0.85)
100(0.05)
99(0.1)
HA
DHA ndc
0.014 ꢁ 0.001 ndc
(R)-1
ndc
ndc
0.33 ꢁ 0.01 0.5 ꢁ 0.1
HA
DHA 0.02 ꢁ 0.01
HA
1.3 ꢁ 0.2
DHA 0.02 ꢁ 0.01
HA
0.9 ꢁ 0.1
DHA 0.26 ꢁ 0.03
HA
2.9 ꢁ 0.5
ndc
4.2 ꢁ 0.3
8.3 ꢁ 0.4
15 ꢁ 1
7(1.0)
7(1.0)
2
3
4
0.28 ꢁ 0.04 3.5 ꢁ 0.4
1.5 ꢁ 0.1 20 ꢁ 2
25(1.0) 56(1.0)
97(0.1) 100(0.1)
8(0.85) 60(0.85)
92(0.1) 93(0.1)
74(0.85) 83(0.3)
86(0.1) 90(0.1)
49 ꢁ 3
0.13 ꢁ 0.03 0.48 ꢁ 0.03 2.1 ꢁ 0.2
0.77 ꢁ 0.02 3.2 ꢁ 0.3
10 ꢁ 1
0.9 ꢁ 0.1
1.2 ꢁ 0.2
9.0 ꢁ 0.2
2.4 ꢁ 0.3
12.0 ꢁ 1.0
38 ꢁ 3
96(0.05)
a
[DHA] or [HA] (100 mM) and acceptor aldehydes (80 mM) total volume 300 mL. Different amounts of enzyme were used for obtaining a linear
dependence of the concentration versus time between 0% and 10% of conversion. Values are the mean of four independent determinations ꢁ
b
standard error. Product formation measured by HPLC after 24 h using an external standard method. Values are the mean of two independent
determinations (see ESIw); percentage calculated with respect to the limiting acceptor aldehyde substrate. c nd: not detected (i.e. r0.01 mmol minꢀ1 mgꢀ1
)
or no peak of the aldol adduct detected by HPLC (detection limit: 0.2–0.5 nmol).
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 5762–5764 5763

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app
app
Table 2 Apparent Vmax
and Km
values of DHA and 4 for their aldol addition reaction
FSA catalysts
Substrate
Vmaxapp/mmol min–1 mg–1
Kmapp/mM
103 ꢂ Vmaxapp/Kmapp/minꢀ1 mgꢀ1
17
47
50
1733
27
1000
333
1533
Wild-type
A129S
A165G
A129S/A165G
Wild-type
A129S
4a
4
4
4
DHA
DHA
DHA
DHA
0.8 ꢁ 0.1
1.9 ꢁ 0.2
1.9 ꢁ 0.2
12 ꢁ 1
169 ꢁ 19b
139 ꢁ 24b
122 ꢁ 15c
24 ꢁ 5c
58 ꢁ 6
0.46 ꢁ 0.02
2.2 ꢁ 0.1
2.2 ꢁ 0.2
15 ꢁ 2
7 ꢁ 2
A165G
A129S/A165G
22 ꢁ 6
32 ꢁ 13
a
b
c
Concentration of 4 could not be higher than 200 mM due to the limited solubility in buffer/DMF 4 : 1 mixtures. Max [4] = 200 mM. Max [4] =
140 mM.
protein residues (Fig. 1B). Furthermore, the models also
suggest that the mutated S129 residue can establish hydrogen
bonds with two of the oxygen atoms of the DHA moiety. This
could increase the nucleophilicity of the reactive carbon of
DHA, which would explain the increased activity observed for
enzymes that contain such mutation. Remarkably good results
were also obtained with the HA donor, which could not be
anticipated since FSA wild-type and the A129S mutant
showed similar velocities for this nucleophile. Furthermore,
the initial reaction velocities (no) supported evidence of the
synergistic effect of the double mutation on FSA. As shown in
Table 1, the improved activity of the double mutation was
4- to 10-fold higher than that of individual mutations for
DHA and 3-fold higher than those for HA.
and 7 (from 40 to 82%) (see ESIw). Furthermore, gaining insight
into identifying the critical residues for the acceptor-binding site of
FSA led to such improved catalytic efficiency. The structure-
guided approach based on site-directed mutagenesis can be, in this
case, a potent tool for further re-designs of FSA and related
aldolases to suit any future specific targets. Work towards this
direction is currently in progress in our lab.
This work was supported by the Spanish MICINN CTQ2009-
07359 and CTQ2009-08328, Generalitat de Catalunya (2009
SGR 00281), and ESF COST CM0701 projects. M. Gutierrez
acknowledges MICINN for the predoctoral scholarship.
Notes and references
z Efforts to improve the yields by optimizing the reaction conditions
resulted unfruitful.
Importantly, the results indicate that the stereochemical
outcome of the FSA mutants towards the selected substrates
was identical to that of FSA wild-type, therefore the mutations
did not change the preferential orientation of the selected
donor and acceptor substrates at the FSA active site (see
ESIw). To gain insight into the catalytic properties of FSA
wild-type and mutants, the apparent steady state kinetic
parameters for the nucleophile DHA and acceptor 4 were
1 M. Schurmann and G. A. Sprenger, J. Biol. Chem., 2001, 276,
¨
11055; M. Schurmann, M. Schurmann and G. A. Sprenger, J. Mol.
¨
¨
Catal. B: Enzym., 2002, 19, 247; J. A. Castillo, J. Calveras, J. Casas,
M. Mitjans, M. P. Vinardell, T. Parella, T. Inoue, G. A. Sprenger,
J. Joglar and P. Clapes, Org. Lett., 2006, 8, 6067; M. Sugiyama,
´
Z. Hong, P. H. Liang, S. M. Dean, L. J. Whalen, W. A. Greenberg
and C.-H. Wong, J. Am. Chem. Soc., 2007, 129, 14811; M. Rale,
S. Schneider, G. A. Sprenger, A. K. Samland and W.-D. Fessner,
Chem.–Eur. J., 2011, 17, 2623.
determined (Table 2).
As expected, in terms of Vmaxapp/Km
2 A. L. Concia, C. Lozano, J. A. Castillo, T. Parella, J. Joglar and
app
the double mutant
P. Clape
3 X. Garrabou, J. A. Castillo, C. Gue
M. Lemaire and P. Clapes, Angew. Chem., Int. Ed., 2009, 48, 5521.
4 J. A. Castillo, C. Guerard-Helaine, M. Gutierrez, X. Garrabou,
M. Sancelme, M. Schurmann, T. Inoue, V. Helaine,
F. Charmantray, T. Gefflaut, L. Hecquet, J. Joglar, P. Clapes,
´
s, Chem.–Eur. J., 2009, 15, 3808.
´
rard-He
´
laine, T. Parella, J. Joglar,
functions with aldehyde 4 B100-fold better than the FSA wild-
app
´
type, mainly due to a B15-fold increase in Vmax
and a
´
´
´
B7-fold decrease in Kmapp. Moreover, as judged by the
Vmaxapp/Kmapp, the FSA A129S/A165G functions with the
nucleophile, DHA, 50-fold better than the FSA wild-type. It
is noteworthy that FSA A129S has still better Kmapp for DHA,
in good agreement with previous observations,4 but lower
´
¨
´
G. A. Sprenger and M. Lemaire, Adv. Synth. Catal., 2010, 352, 1039.
5 R. J. Molyneux, Y. T. Pan, J. E. Tropea, A. D. Elbein, C. H. Lawyer,
D. J. Hughes and G. W. J. Fleet, J. Nat. Prod., 1993, 56, 1356;
N. Asano, K. Yasuda, H. Kizu, A. Kato, J.-Q. Fan, R. J. Nash,
G. W. J. Fleet and R. J. Molyneux, Eur. J. Biochem., 2001, 268, 35;
K. Yasuda, H. Kizu, T. Yamashita, Y. Kameda, A. Kato, R. J. Nash,
G. W. J. Fleet, R. J. Molyneux and N. Asano, J. Nat. Prod., 2002, 65,
198; Y. F. Wang, D. P. Dumas and C.-H. Wong, Tetrahedron Lett.,
1993, 34, 403.
app
app
Vmax
A129S/A165G compensates the Km
the best mutant for DHA. The Vmaxapp/Km
than the double mutant. The higher Vmax
of FSA
app
value turning out to be
app
values for
aldehyde 4 imply that the double mutant is much more efficient
in transforming this substrate than the FSA wild-type. The
results obtained may be extrapolated to the other selected
substrates and are consistent with the fact that the best catalyst
for the aldol addition reactions of DHA to aldehydes (S)-1,
(R)-1, 2, and 3 is the FSA A129S/A165G double mutant.
To sum up, the FSA A129S/A165G double mutant constitutes
a promising biocatalyst for syn configured aldol reactions of
DHA and HA with wider acceptor aldehyde selectivity as com-
pared with FSA wild-type. Examples of isolated yields of aldol
adducts improved significantly as compared with FSA wild-type
and RAMA: (5S)-5 (from 10 to 83%), (5R)-5 (from 46 to 72%)
6 L. Espelt, T. Parella, J. Bujons, C. Solans, J. Joglar, A. Delgado and
P. Clapes, Chem.–Eur. J., 2003, 9, 4887.
´
7 G. W. J. Fleet, S. J. Nicholas, P. W. Smith, S. V. Evans, L. E. Fellows
and R. J. Nash, Tetrahedron Lett., 1985, 26, 3127; G. W. J. Fleet and
P. W. Smith, Tetrahedron, 1986, 42, 5685; J. P. Saludes, S. C. Lievens
and T. F. Molinski, J. Nat. Prod., 2007, 70, 436; B. Andersen,
A. Rassov, N. Westergaard and K. Lundgren, Biochem. J., 1999,
342, 545; K. Fosgerau, N. Westergaard, B. Quistorff, N. Grunnet,
M. Kristiansen and K. Lundgren, Arch. Biochem. Biophys., 2000, 380,
274; A. B. Walls, H. M. Sickmann, A. Brown, S. D. Bouman,
B. Ransom, A. Schousboe and H. S. Waagepetersen, J. Neurochem.,
2008, 10, 1.
8 S. Schneider, M. Gutierrez, T. Sandalova, G. Schneider, P. Clapes,
´ ´
G. A. Sprenger and A. K. Samland, ChemBioChem, 2010, 11, 681.
c
5764 Chem. Commun., 2011, 47, 5762–5764
This journal is The Royal Society of Chemistry 2011