Complete Switch of Reaction Specificity of an Aldolase by Directed Evolution In Vitro: Synthesis of Generic Aliphatic Aldol Products

Article abstract of DOI:10.1002/anie.201804831

A structure-guided engineering of fructose-6-phosphate aldolase was performed to expand its substrate promiscuity toward aliphatic nucleophiles, that is, unsubstituted alkanones and alkanals. A “smart” combinatorial library was created targeting residues D6, T26, and N28, which form a binding pocket around the nucleophilic carbon atom. Double-selectivity screening was executed by high-performance TLC that allowed simultaneous determination of total activity as well as a preference for acetone versus propanal as competing nucleophiles. D6 turned out to be the key residue that enabled activity with non-hydroxylated nucleophiles. Altogether 25 single- and double-site variants (D6X and D6X/T26X) were discovered that show useful synthetic activity and a varying preference for ketone or aldehyde as the aldol nucleophiles. Remarkably, all of the novel variants had completely lost their native activity for cleavage of fructose 6-phosphate.

Full text of DOI:10.1002/anie.201804831

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Accepted Article
Title: Complete switch of reaction specificity of an aldolase by directed
evolution in vitro: Synthesis of generic aliphatic aldol products
Authors: Wolf-Dieter Fessner, Sebastian Junker, Raquel Roldan,
Henk-Jan Joosten, and Pere Clapés
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201804831
Angew. Chem. 10.1002/ange.201804831
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http://dx.doi.org/10.1002/ange.201804831

Angewandte Chemie International Edition
10.1002/anie.201804831
COMMUNICATION
Complete switch of reaction specificity of an aldolase by directed
evolution in vitro: Synthesis of generic aliphatic aldol products
[
a]
[b]
[c]
[b]
[a]
Sebastian Junker, Raquel Roldan, Henk-Jan Joosten, Pere Clapés and Wolf-Dieter Fessner*
Abstract: A structure-guided engineering of fructose-6-phosphate
aldolase was performed to expand its substrate promiscuity toward
aliphatic nucleophiles, i.e., unsubstituted alkanones and alkanals. A
synthesis, however, products from unsubstituted aliphatic
nucleophiles (simple ketones and aldehydes) could only be
isolated when applying L-GA3P as the electrophile with catalysis
by FSA(D6H), presumably because phosphorylation confers
high substrate binding affinity.
“
smart” combinatorial library was created targeting residues D6, T26
and N28 that form a binding pocket around the nucleophilic carbon
atom. Double-selectivity screening was executed by high-
performance TLC that allowed simultaneous determination of total
activity as well as a preference for acetone versus propanal as
competing nucleophiles. D6 turned out to be the key residue that
enabled activity with non-hydroxylated nucleophiles. Altogether 25
single- and double-site variants (D6X and D6X/T26X) were
O
O
OH O
wt-FSA
OH
OH
2-
+
+
2-
O PO
O PO
3
3
OH
OH
OH OH
D-GA3P
DHA
D-Fru6P
O
O
OH O
discovered that show useful synthetic activity and
a varying
FSA (D6H)
2-
2-
O PO
O PO
3
preference for ketone or aldehyde as the aldol nucleophiles.
Remarkably, all of the novel variants had completely lost their native
activity for cleavage of fructose 6-phosphate.
3
OH
OH
L-GA3P
1
L-1,3-dideoxy-
Sor6P
O
O
OH
O
wt-FSA
wt-FSA
Aldolases and transaldolases are specialized on sugar
phosphates, yet highly interesting biocatalytic tools for chemical
synthesis because of their precise stereoselectivity in the
carboligation step and their activity under very mild reaction
+
+
2
1
3
O
O
OH
O
OH OH O
wt-FSA
[
1,2]
conditions.
For example, fructose-6-phosphate aldolase
+
2
2
2
4
5
(
FSA) from E. coli is a thermostable Class I enzyme, which
Scheme 1. Native use of DHA nucleophile by wild-type FSA and promiscuous
reversibly catalyses the highly stereoselective addition of
dihydroxyacetone (DHA) to D-glyceraldehyde 3-phosphate (D-
GA3P) resulting in the formation of D-fructose 6-phosphate (D-
tolerance for acetone of the D6H variant with phosphorylated aldehyde
[
8]
electrophile. Catalyzed addition of unsubstituted nucleophiles (acetone,
propanal) to generic aliphatic electrophiles is yet unknown.
[
3-5]
Fru6P; Scheme 1).
Like other aldolases, FSA accepts a
broad range of aldehydes as the electrophilic aldol
[
2,5,6]
component.
However, while most aldolases are quite
An aldolase having the ability to utilize generic aliphatic, non-
hydroxylated substrates both as electrophilic and nucleophilic
components would constitute a highly flexible catalyst for a
plethora of synthetic opportunities toward the construction of
chiral building blocks by Green Chemistry principles. The range
of addressable targets from such reactions, such as the homo
aldol products of aldehydes (e.g., 4, 5), are common structural
specific for their nucleophilic substrate, FSA tolerates some
structural variation of the DHA nucleophile and also converts
[
6]
hydroxyacetone, hydroxybutanone and hydroxyethanal.
Recently, we demonstrated that designed minimal mutations in
the FSA active site could further open up the nucleophile
tolerance for large ketols at least up to the size of 1-
[
7]
[9]
[10]
hydroxyheptan-2-one. Lifting the absolute requirement for α-
hydroxylation in the ketol nucleophiles, residual activity with
generic aliphatic ketone substrates could be detected for wild-
type FSA and its D6H variant by using a fluorogenic assay
motifs in natural products like polyketides or terpenoids, but
are also important as intermediates in the production of
[
11]
pharmaceuticals
and industrial bulk chemicals such as the
[
12]
Guerbet-type compounds.
Here we present the engineering
[
8]
by a directed evolution approach of novel FSA variants, which
selectively use acetone (1) or propanal (2) as nucleophilic
substrates with high carboligation stereoselectivity.
principle in aldol cleavage direction. In direction of aldol
From an inspection of the enzyme active site, using the X-
ray structure of FSA with a model of the D-Fru6P substrate
[
a]
S. Junker, Prof. Dr. W.-D. Fessner
Institut für Organische Chemie und Biochemie
Technische Universität Darmstadt
[
8]
bound to the catalytic lysine (K85), it is evident that the 3-OH
group in the nucleophile moiety interacts with residue D6 via
hydrogen bonding and loosely contacts the backbone amide
group of N28 (Fig. 1). D6 also forms a hydrogen bond with 5-OH
of Fru6P. In addition, T26 donates a hydrogen bond to D6,
which aligns it for an optimal substrate interaction. Replacing
these polar residues by hydrophobic ones would interrupt the
stabilizing contacts and thereby should improve an alternative
binding of non-hydroxylated nucleophiles such as acetone or
Alarich-Weiss-Str. 4, 64287 Darmstadt, Germany
E-mail: fessner@tu-darmstadt.de
[
[
b]
c]
Raquel Roldan, Prof. Dr. Pere Clapés
Instituto de Quım ́i ca Avanzada de Cataluña-IQAC-CSIC, Jordi
Girona 18-26, 08034 Barcelona, Spain
Dr. Henk-Jan Joosten
Bio-Prodict, Nieuwe Marktstraat 54e, 6511 AA Nijmegen, The
Netherlands
Supporting information for this article is given via a link at the end of
the document.
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Angewandte Chemie International Edition
10.1002/anie.201804831
COMMUNICATION
aliphatic aldehydes. The side chain of N28, however, donates a
hydrogen bond to the 4-OH group, which stabilizes the incipient
oxyanion formation at the aldehyde carbonyl group upon aldol
Library screening was performed with whole cells in aqueous
medium containing a mixture of acetone and propanal (1:2 =
17:1, v:v) as competitive substrates. Although the aldehyde was
essential to serve as the electrophile, the ketone proportion was
strongly increased to compensate for its lower reactivity with the
catalytic lysine. Samples were periodically analyzed by TLC for
product formation. Roughly 55% of the clones yielded one or two
new product spots (Figure 2) with intensities stronger than wild-
type, whereas others barely produced detectable product
amounts or were inactive.
[
13]
attack and render mutation of N28 problematic.
Figure 2. Exemplary TLC analysis using acetone and propanal as substrates
catalysed by hit plate candidates having different activity and nucleophile
selectivity. Anisaldehyde staining reveals a blue spot corresponding to the
homo-aldol 4, and a green spot for the cross-aldol 3. Lanes correspond to
variant sequences A = D6H, B = D6H/T26L, C = D = D6A/T26A (duplicate
clone), E = T26I, F = wt, G = D6L/T26I, H = D6P/T26L. Image contrast was
digitally improved for visualization.
[
15]
Figure 1. PyMOL
model of the active site of the wild-type FSA from E.
[4]
[
8[
coli. The X-ray structure of FSA (PDB entry 1l6w) was aligned with the D-
[
14]
F6P liganded in a highly similar transaldolase complex (PDB entry 3s1v).
The “essential” 3-OH group is highlighted by an arrow.
Re-screening and sequencing furnished a total set of 25 unique
genetic variants. The latter were further characterized for their
relative kinetic activities and for their substrate selectivity against
To limit the screening efforts we tested individual rational
replacements for a first orientation. Indeed, single variants D6A,
1
and 2 by automated high-performance TLC (HPTLC) analysis
[
8]
D6L, D6E and D6H were found to be active with acetone as
nucleophile and GA3P as electrophile. Exploratory saturation
N28X in FSA variants D6E, D6H and D6L abolished activity.
Thus, simultaneous site saturation mutagenesis was
performed on the two remaining sites. To reduce the library to a
manageable size a smart construction approach was followed by
using densitometrical product quantification. We found that
HPTLC is a medium-throughput screening method, which is well
suited for the direct screening of aldol product formation,
allowing for simultaneous analysis of both activity and selectivity.
It is complementary to the typical inverse determination of
[3]
aldolase activity by aldol cleavage assays albeit limited to
[
16]
eliminating mutations unlikely to yield positive hits.
structure-based sequence alignment for the aldolase
A
endpoint measurements. The HPTLC method was validated by
a comparison to standard GC results, using reactions catalyzed
by FSA variants D6E, D6L, D6A and D6L/T26A.
[
17]
superfamily from Bio-prodict (3DM database)
delivered the
amino acid distribution at positions that are structurally
conserved within the whole superfamily. By encoding only the
Among the 25 unique hits, 16 (64%) showed a clear
competitive preference for one over the other nucleophile
3
DM-derived subset instead of all 20 proteinogenic amino acids
(
nucleophile preference >3:1), while the other 9 (36%) produced
the library size can be significantly reduced. While the D6/T26
motif is highly conserved, an analysis of the correlated mutation
both products 3 and 4 in similar quantities (Scheme 2). The 16
selective variants were further analyzed by GC for an accurate
determination of their nucleophile selectivity (Figure 3). The
analysis shows that the two positions (D6/T26) selected for
mutagenesis are indeed the expected hot spots for changing the
catalytic properties of the enzyme, and that the “smart library”
strategy paid off with a very high proportion of positive hits (25
out of 48 library members, 52% hit rate). The conditions chosen
for substrate competition facilitated a direct evaluation of both
activity and nucleophile selectivity, which could be read out
using conventional medium-throughput TLC screening.
[
18]
data
of the 3DM database showed that there is a functional
connectivity between the amino acids. Variations occur only at
very low frequency, e.g., for the entire Tal family A/I 19.85%; A/L
9
0
.25%; D/A 0.27%, and for FSA-type enzymes L/T 0.74%; D/C
.56%; D/D, D/S, P/T, R/T each 0.19%. Therefore, we created a
combinatorial library where Asp6 was mutated by using the
restricted SHH set coding for A, D, Q, E, H, L, P, and V, and T26
by using the VYT set coding for A, I, L, P, T and V (8 x 6 = 48
variants requiring the screening of 200 clones for 95%
[
19]
coverage).
Also, unusual solutions revealed by the 3DM
Clearly, non-hydroxylated nucleophiles become favored if the
hydrogen-bonding pattern directed at the 3-OH group are
deleted via non-polar D6 replacement (D6A, D6V, D6L, D6P), or
software tool, such as uncommon but allowed variants D6A, D6L
and D6P as well as the D6A/T26I and D6A/T26L combinations,
are included in this setting.
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Angewandte Chemie International Edition
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COMMUNICATION
if mutant residues are incompatible with the hydrogen-bonding
network and occupy an additional volume (D6H, D6E, D6Q).
While under the specific conditions many active variants
appeared rather non-selective (e.g., D6E), some variants
showed very high complementary selectivity for either of the
aldehyde or ketone nucleophiles. A detailed analysis of the
The very high selectivity of several variants to utilize 2 as
nucleophile was unexpected, given its low concentration in
comparison to excessive 1. Separate experiments were
conducted to determine relative catalytic activities in the
absence of nucleophile competition, firstly by using 2 as the sole
substrate (Figure 4, left); as a reference enzyme we used the
[
8]
[20]
selective candidates revealed that the known D6H variant was
the most selective catalyst for 1, similar to the higher active D6L
variant (Figure 3). However, most of the selective variants
preferred the more reactive 2 as nucleophile, despite of its much
lower assay concentration. Interestingly, the primary preference
introduced by the D6X replacement can be strongly shifted
DERA from E. coli (F200I variant ), which so far is the only
[
21]
enzyme known to accept 2 as a non-native nucleophile.
A
complementary screen was performed by using in the
1
presence of an aldehyde electrophile (6) that itself is unreactive
for self aldolization (Figure 4, right).
towards improved preference for
particularly with residues having increasing hydrophobic volume
A<<V<L<I). The effect is observed for D6E, D6L, D6H, and is
particularly effective for D6H, where strong switch of
2
by T26X exchanges,
FSA
D6X/T26X)
O
O
O
O
O
O
OH
O
O
OH O
(
(
+
+
+
(
2
6
8
1
3
4
a
preference from 1 (D6H) to 2 (D6H/T26L) takes place, which
both show similar rates. A synonymous trend is seen for D6A or
D6P but to a lesser extent because these are already quite
selective for 2. While the T26V and T26I are mostly
accompanied by lower rates, the T26L mutants typically show
FSA
D6X/T26X)
OH
O
1
7
FSA
D6X/T26X)
OH
O
O
(
higher rate and thus represent the best choice with
a
+
(
compromise in rate and selectivity. The highest selectivity for 2
was determined for the D6Q/T26I variant but only at a rather low
rate. Surprisingly, the simple T26L mutation alone results in
significantly increased rate and selectivity for 2. Notably, proline
is tolerated in the D6 position but is not tolerated to replace T26;
usually, introduction of proline is cumbersome because of
conformational restrictions.
1
9
FSA
D6A/T26I)
OH
O
O
O
i), ii)
i), ii)
2
4
10
FSA
(D6A/T26I)
OH
O
O
O
11
12
13
Scheme 2. Reactions used for screening the nucleophile preference of the
D6X/T26X library. Stereoselectivity was analyzed via cyclic acetal formation.
+
Reaction conditions: i) NaBH
4
, MeOH ii) 2,2-dimethoxypropane, H .
Figure 4. Blue bars: rate of formation of 4 by self-aldolization of 2 catalyzed by
FSA variants most active in primary screenings; wt-FSA and DERA from E.
coli (light blue) were included for reference. Green bars: rate of formation of 11
by addition of 1 to 6 using FSA variants most active on 1. For reaction
conditions see Supporting Info.
In the first tests, most active FSA variants, independent of their
nucleophile selectivity, were screened for their rate of formation
of 4. The concentration of 2 had to be limited (83 mM) because
of its denaturation effect on the enzyme at higher substrate
concentrations. Whereas wild-type FSA showed barely any
Figure 3. Activity and competitive nucleophile bias of selective FSA variants,
in comparison to wild-type and the D6E variant as a non-selective example.
Relative cumulative rates (GC peak sum for products 3 + 4) are based on
most active variant D6L. Green bars indicate preference for 1 (major product
3
) as nucleophile, blue bars for 2 (major product 4).
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COMMUNICATION
detectable product formation, several variants displayed good
activity. Several new FSA variants (D6A/T26A, D6A/T26V,
D6E/T26A) were found to have activity superior to the DERA
reference. A distinctive feature is that reactions catalyzed by
FSA variants stop after the first addition to form 4, while DERA
catalyzes a consecutive addition to give a trimer of type 5, which
and to the straight-chain isomer 8, yielding aldol products 7 and
9, respectively.
(
2S)-Configuration can be anticipated for steric reasons
when using 2 as a nucleophile; however, as this was never
[
21]
rigorously proven for the corresponding DERA reaction, there
was no reference available. Thus, product 4 obtained on
preparative scale (21% yield) was converted into the cyclic
derivative 10 for stereochemical analysis (Scheme 2). NMR
analysis clearly demonstrated the cis-(2S,3R) arrangement of
the alkyl substituents, and chiral GC analysis showed the
presence of only a single enantiomer (>99% ee). As a control,
aldehyde 4 was exposed to alkaline conditions before being
[
21]
can cyclize into a lactol form.
In the second test series, variants selective for 1 were
investigated for their relative rates. As the electrophile,
isopentanal (6) was employed when isobutanal proved
unreactive with common nucleophiles; both did not show any
reactivity for self-aldolization even at high enzyme
concentrations, probably because of their steric bulkiness. The
corresponding aldol products are acyclic and generated by a
bimolecular synthetic process, thus thermodynamically
processed, which yielded
a
mixture of cis and trans
[
25]
diastereomers.
Thus, the native (3S,4R)-stereoselectivity of
[
22]
the wild-type FSA was fully retained with non-hydroxylated,
hydrophobic substrate analogs. Gratifyingly, FSA variants
D6A/T26L and D6A/T26I were even active with butanal (11) to
form the aldol dimer 12 with about one third of the rate for 4 and
with the same diastereoselectivity, as proven by a corresponding
analysis of the derivative 13. The larger homolog 8 did not lead
to aldol formation. Both compounds 4 and 12 are formal
precursors to industrially important Guerbet-type compounds,
unfavorable,
but acetone can be used at rather high
concentration to drive the equilibrium towards product formation
because FSA is quite stable to organic cosolvents. Screening for
the formation of 7 under these non-competitive conditions
showed that only the single mutants D6L and D6H have highest
activity, whereas mutant combinations caused lower rates.
Unfortunately, for none of the reactions studied we could find
[
12]
a
reliable in vitro assay to perform steady-state kinetic
which are used as bulk solvents/plasticizers. It is tempting to
speculate that enzymatic routes to such man-made bulk
measurements with purified enzymes in direction of synthesis.
Probably owing to its low electrophilic nature, acetone has only
[
26]
materials
could be realized using FSA-derived catalysts in a
M
low binding affinity to FSA and no K could be estimated. Clearly, biotechnology process.
rate differences reported are not due to different expression
levels, which were checked by SDS-PAGE, nor to different
protein stabilities, which were checked by differential scanning
In summary, following a single smart library strategy it was
possible to identify a collection of new FSA variants having novel
activities and distinct preferences for the nucleophilic substrate,
and that are promising for possible use in the stereospecific
synthesis of chiral building blocks. These enzymes significantly
expand the product space hitherto addressable by enzymatic
[
7]
fluorimetry.
The novel activities and selectivities most likely are due to an
improved binding of hydrophobic nucleophiles because the main
catalytic mechanism of the Class I aldolase, involving covalent
Schiff base formation at K85 and tyrosine residues acting as an
[
1,2]
carboligation.
Remarkably, the synthesis of all generic aldol
products such as 3, 4, 7, 9 or 12 — stripped from the abundant
polar functionalization of native substrates — could also be
performed conveniently using recombinant whole cell catalysis
because such substrates and products seem to permeate quite
well across the cell wall. It is appropriate to stress that all new
enzyme variants completely lost the native FSA activity for
cleavage of Fru6P, nor do they show residual synthetic activity
with hydroxylated nucleophiles.
[
22,23]
acid-base catalyst,
remains intact. A major effect is caused
by replacements of D6 that interrupt the hydrogen-bonding
network for recognition of the 3-OH group and occupy a larger
space than the native aspartate (D6L, D6H, D6E, D6Q; except
for D6A). Especially, D6H and D6L show good activity with 1,
because the compact remaining space seems inappropriate to
bind the larger alkyl moiety of 2. Considering the large excess of
1
in the assay solution, the collective data indicate that all other
This study shows that
a complete switch in reaction
variants essentially have a preference for 2, which can be further
enhanced by hydrophobic replacements of T26. Apparently, the
side chain volumes of T26V, T26I and T26L restrict the most
distant cavity space of the active site where C1 of the substrate
is located. Thereby, binding of the C1 moiety of a ketone
nucleophile becomes obstructed in favor of a smaller aldehyde
moiety. Among the positive T26 variants the T26L mutation
seems to be the most adaptive, while T26I needs to be
combined with D6A replacement to compensate for its steric
bulkiness.
specificity of an aldolase can be realized by focusing directed
evolution on a confined hot spot of substrate binding. By
exploiting existing knowledge on enzyme structure-function
relationships and exhaustive bioinformatic analysis of protein
databases, smart combinatorial libraries can be rapidly designed
that offer very high hit rates. We could also show that parallel,
automated HPTLC analysis is a valuable medium-throughput
screening tool that is well suited for such a highly focused
approach. We believe that the direct monitoring of product
formation is more flexible, and furnishes more appropriate data
for an evaluation of the synthetic capacity of carboligation
catalysts, as compared to the conventional indirect monitoring of
fragments arising from aldol cleavage.
Stereoselectivity is a critical issue when the electrophile C=O
[
5]
is not firmly positioned and can be attacked from both faces.
However, product 3 obtained on a preparative scale (34% yield)
[
24]
showed high enantiomeric purity (ee >95%) by chiral GC.
Similar results were obtained by preparative addition of 1 to 6
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Angewandte Chemie International Edition
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COMMUNICATION
[
18] T. van den Bergh, G. Tamo, A. Nobili, Y. Tao, T. Tan, U. T.
Bornscheuer, R. K. P. Kuipers, B. Vroling, R. M. de Jong, K.
Subramanian, P. J. Schaap, T. Desmet, B. Nidetzky, G. Vriend, H.-J.
Joosten, PLoS One 2017, 12, e0176427.
Experimental Section
For experimental details, see Supporting Info..
[
[
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20] S. Jennewein, M. Schuermann, M. Wolberg, I. Hilker, R. Luiten, M.
Wubbolts, D. Mink, Biotechnol. J. 2006, 1, 537.
Acknowledgements
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[
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22] C. Zeymer, R. Zschoche, D. Hilvert, J. Am. Chem. Soc. 2017, 139,
This project received funding from the European Union’s
Horizon 2020 research and innovation program under grant no.
1
2541-12549.
[
23] V. Sautner, M. M. Friedrich, A. Lehwess-Litzmann, K. Tittmann,
Biochemistry 2015, 54, 4475-4486.
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35595 (CarbaZymes), the Spanish Ministerio de Economía y
Competitividad (MINECO), and the Fondo Europeo de
Desarrollo Regional (FEDER) (grant no. CTQ2015-63563-R to
P.C.).
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Keywords: biocatalysis • fructose-6-phosphate aldolase •
HPTLC screening • protein engineering • stereoselectivity
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Angewandte Chemie International Edition
10.1002/anie.201804831
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
A “smart library” strategy identified
D6/T26 as the key residues in
fructose-6-phosphate aldolase (FSA)
to expand its substrate promiscuity
toward unsubstituted alkanones and
alkanals as non-natural nucleophiles.
Double-selectivity HPTLC screening
allowed simultaneous determination
of total activity as well as preference
for competing nucleophiles. Remark-
ably, all of the novel variants had
completely lost their native activity for
cleavage of fructose 6-phosphate.
S. Junker, R. Roldan, H.-J. Joosten, P.
Clapés and W.-D. Fessner *
Page No. – Page No.
Complete switch of reaction
specificity of an aldolase by directed
evolution in vitro: Synthesis of
generic aliphatic aldol products
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