Engineering a Promiscuous Tautomerase into a More Efficient Aldolase for Self-Condensations of Linear Aliphatic Aldehydes

Article abstract of DOI:10.1002/cbic.201700121

The enzyme 4-oxalocrotonate tautomerase (4-OT) from Pseudomonas putida mt-2 takes part in a catabolic pathway for aromatic hydrocarbons, where it catalyzes the conversion of 2hydroxyhexa-2,4-dienedioate into 2-oxohexa-3-enedioate. This tautomerase can also promiscuously catalyze carbon–carbon bond-forming reactions, including various types of aldol reactions, by using its amino-terminal proline as a key catalytic residue. Here, we used systematic mutagenesis to identify two hotspots in 4-OT (Met45 and Phe50) at which single mutations give marked improvements in aldolase activity for the self-condensation of propanal. Activity screening of a focused library in which these two hotspots were varied led to the discovery of a 4-OT variant (M45Y/F50V) with strongly enhanced aldolase activity in the self-condensation of linear aliphatic aldehydes, such as acetaldehyde, propanal, and butanal, to yield α,β-unsaturated aldehydes. With both propanal and benzaldehyde, this double mutant, unlike the previously constructed single mutant F50A, mainly catalyzes the self-condensation of propanal rather than the cross-condensation of propanal and benzaldehyde, thus indicating that it indeed has altered substrate specificity. This variant could serve as a template to create new biocatalysts that lack dehydration activity and possess further enhanced aldolase activity, thus enabling the efficient enzymatic self-coupling of aliphatic aldehydes.

Full text of DOI:10.1002/cbic.201700121

Accepted Article
Title: Engineering a Promiscuous Tautomerase into a More Efficient
Aldolase for Self-Condensations of Linear Aliphatic Aldehydes
Authors: Mehran Rahimi, Jan Ytzen van der Meer, Edzard Geertsema,
and Gerrit Jan Poelarends
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To be cited as: ChemBioChem 10.1002/cbic.201700121
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10.1002/cbic.201700121
ChemBioChem
Engineering a Promiscuous Tautomerase into a More Efficient Aldolase for
Self-Condensations of Linear Aliphatic Aldehydes
Mehran Rahimi,[a] Jan-Ytzen van der Meer,[a] Edzard M. Geertsema,[a] and
Gerrit J. Poelarends*[a]
[a]
Dr. M. Rahimi,+ Dr. J.-Y. van der Meer, + Dr. E.M. Geertsema,‡ Prof. Dr. G.J. Poelarends
Department of Chemical and Pharmaceutical Biology
Groningen Research Institute of Pharmacy, University of Groningen
Antonius Deusinglaan 1, 9713 AV Groningen (The Netherlands)
E-mail: g.j.poelarends@rug.nl
[+]
These authors contributed equally to this work
[‡]
Present address: Institute for Life Science & Technology, Hanze University of Applied
Sciences, Zernikeplein 11, 9747 AS Groningen
Keywords:
4-oxalocrotonate tautomerase, catalytic promiscuity, aldolase activity,
condensation reaction, protein engineering
1
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ABSTRACT
The enzyme 4-oxalocrotonate tautomerase (4-OT) from Pseudomonas putida mt-2 takes part in a
catabolic pathway for aromatic hydrocarbons, where it catalyzes the conversion of 2-
hydroxyhexa-2,4-dienedioate into 2-oxohexa-3-enedioate. This tautomerase can also
promiscuously catalyze carbon-carbon bond-forming reactions, including various types of aldol
reactions, utilizing its amino-terminal proline as a key catalytic residue. Here, we have used a
systematic mutagenesis strategy to identify two ‘hotspot’ positions in 4-OT (Met-45 and Phe-50)
at which single mutations give a marked improvement in aldolase activity for the self-
condensation of propanal. Activity screening of a focused library in which these two ‘hotspot’
positions were varied led to the discovery of a 4-OT variant (M45Y/F50V) with strongly
enhanced aldolase activity in the self-condensation of linear aliphatic aldehydes, such as
acetaldehyde, propanal and butanal, yielding α,β-unsaturated aldehydes. In the presence of both
propanal and benzaldehyde, this double mutant, unlike the previously constructed single mutant
F50A, mainly catalyzes the self-condensation of propanal rather than the cross-condensation of
propanal and benzaldehyde, indicating that it indeed has an altered substrate specificity. This
M45Y/F50V variant of 4-OT could serve as a starting template to create new biocatalysts that
lack dehydration activity and possess further enhanced aldolase activity, enabling the efficient
enzymatic self-coupling of aliphatic aldehydes.
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INTRODUCTION
The enzyme 4-oxalocrotonate tautomerase (4-OT) is composed of six identical subunits of only
62 amino acid residues each. It is a member of the tautomerase superfamily, a group of
homologous proteins having a β-α-β structural fold and a catalytic amino-terminal proline (Pro-1)
in common.[1-3] 4-OT takes part in a catabolic pathway for aromatic hydrocarbons in
Pseudomonas putida mt-2, where it catalyzes the conversion of 2-hydroxyhexa-2,4-dienedioate
(1, Scheme 1) into 2-oxohexa-3-enedioate (2).[4,5] In this tautomerization reaction, Pro-1 acts as a
general base (pKa of Pro-1 ~6.4) abstracting the 2-hydroxyl proton of 1 and transferring it to the
C5-position to give 2.[6]
Scheme 1. Proton-transfer reaction naturally catalyzed by 4-OT.
In addition to its natural tautomerase activity, 4-OT has been shown to promiscuously
catalyze several carbon-carbon bond-forming reactions.[7a, 8-16] These include various types of
aldol reactions such as the self-condensation of propanal (3 in Scheme 2), the cross-condensation
of acetaldehyde (6) with benzaldehyde (12), the cross-coupling of propanal (3) and benzaldehyde
(12), and the intramolecular cyclization of hexanedial (15) or heptanedial.[10,12,16] For the self-
condensation of 3, as well as the cross-condensation of 6 and 12, it has been demonstrated that 4-
3
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OT catalyzes both the initial aldol-coupling step to yield the β-hydroxyaldehyde intermediate and
the subsequent dehydration step to yield the final α,β-unsaturated aldehyde product.[12,16] In the
proposed mechanism for the aldolase activity of 4-OT, the active site Pro-1 residue functions as a
nucleophile, rather than a base, and reacts with the carbonyl functionality of the aldehyde to form
a covalent enamine intermediate.[10,17] This intermediate reacts with another aldehyde in an inter-
or intramolecular aldol addition, after which the final product (the aldol compound or the
corresponding dehydrated compound) is released from the active site upon hydrolysis.
Enzyme promiscuity has great promise as a source of synthetically useful catalytic
transformations,[2,7] and could be exploited as starting point to create new biocatalysts for
challenging aldol reactions.[18,19] In previous work, we have constructed several active site
mutants of 4-OT, including the variant F50A, which possess improved aldolase activity for the
cross-condensation of 6 with 12.[12,20] Here, we report the engineering of 4-OT into a more
efficient aldolase for self-condensation reactions of linear aliphatic aldehydes such as
acetaldehyde (6), propanal (3) and butanal (9) (Scheme 2). For this, a large collection of single
mutants of 4-OT was first screened for single mutations that give a strong improvement in the
desired aldolase activity, after which identified ‘hotspot’ positions were subjected to
combinatorial mutagenesis.
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Scheme 2. Examples of aldol reactions promiscuously catalyzed by 4-OT.
RESULTS AND DISCUSSION
We applied a systematic mutagenesis strategy to identify residue positions at which mutations
give a marked improvement in the aldolase activity of 4-OT. For this, a collection of 1040 single
mutants of 4-OT, covering at least 15 of the 19 possible variants at each residue position (from
Ile-2 to Arg-62), was used.[21] Single mutants of the Pro-1 residue were not included in the
collection, because Pro-1 is a key catalytic residue and mutations at this position lead to incorrect
demethionylation of the protein.[22] The complete collection of 4-OT mutants was screened for
activity for the self-condensation of 3, using conditions that allowed for the detection of variants
with strongly improved aldolase activity only. The results are graphically represented in a
heatmap (Figure 1).
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Figure 1. Heatmap depicting the aldolase activity of single mutants of 4-OT for the self-
condensation of propanal. a, The horizontal axis of the data matrix depicts the wild-type sequence of 4-
OT and the vertical axis depicts the 20 possible amino acid residues. The wild-type amino acid residue at
each position is indicated by bold squares. White boxes represent single mutants which were not present in
the collection, grey boxes represent mutants which were not produced above the detection limit (0.5
mg/ml in the CFE), and boxes with a crosshatch pattern indicate mutants which precipitated under the
screening conditions, preventing activity measurements. The ability of each single mutant of 4-OT to
catalyze the self-condensation of 3 was monitored by UV-spectroscopy, following the formation of 5 (λmax
= 234 nm). b, The secondary-structure elements of 4-OT.
Two residue positions at which single mutations lead to large improvements in the aldolase
activity of 4-OT were identified. The first ‘hotspot’ position is Met-45; replacement of this
residue with Thr, His or Ile resulted in a pronounced improvement in aldolase activity. The
second ‘hotspot’ position is Phe-50; substitution of this residue by Val strongly improved the
aldolase activity of 4-OT. In addition to these beneficial mutations, it was found that the
replacement of His-6 with Met, and the mutation of Ala-33 to Lys, also significantly improved
the aldolase activity of 4-OT. Mutant F50V showed the highest aldolase activity among all
variants. Progress curves of the aldol self-condensation of 3 (50 mM) catalyzed by purified
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enzymes confirmed the enhanced activity of mutant F50V when compared to wild-type 4-OT or
the previously constructed mutant F50A[12] (Figure 2).
Figure 2. UV traces monitoring the formation of 5 during the self-condensation of 3 catalyzed by wild-
type 4-OT (low activity), mutant F50A, mutant F50V, or mutant M45Y/F50V.
To further improve the aldolase activity of 4-OT, we constructed a double-site library in
which the two ‘hotspot positions’ Met-45 and Phe-50 were simultaneously randomized by using
NNK-codon degeneracy,[23,24] covering all 20 possible amino acids, for both positions. In addition
to this double-site library, we constructed a triple-site library in which positions His-6, Met-45,
and Phe-50 were simultaneously randomized. The NNK-codon degeneracy was used for
randomization of positions His-6 and Met-45, whereas Phe-50 was randomized using a NYK-
codon degeneracy.[23,24] The NYK-codon degeneracy was chosen for position Phe-50 because it
reduces the library size by covering only the codons of nine different aliphatic and polar amino
acid residues, including those residues which result in an enhanced aldolase activity, as well as
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the wild-type residue. The two libraries were used to transform Escherichia coli BL21(DE3)
cells. The double-site library was screened by evaluating ~800 transformants, whereas ~3500
transformants of the triple-site library were screened for the aldol self-condensation of 3.
Screening of the double-site library resulted in the identification of a double mutant,
M45Y/F50V, with strongly enhanced aldolase activity (~30-fold improvement compared to wild-
type 4-OT), whereas screening of the triple-site library did not yield a mutant with higher activity
than that of mutant M45Y/F50V. The progress curves of the aldol self-condensation of 3 (50
mM) catalyzed by purified enzymes confirmed the enhanced activity of mutant M45Y/F50V
1
when compared to the single mutants F50V and F50A (Figures 2 and S1). Mutant H NMR
spectroscopic analysis confirmed the formation of 5 as product of the aldol self-condensation of 3
catalyzed by mutant M45Y/F50V (Figure S2). Hence, 4-OT can be engineered into a more
efficient aldolase for the self-condensation of 3 to yield 5 by exploring small libraries in which
only ‘hotspot’ positions are varied.
Having established that mutant M45Y/F50V has improved aldolase activity for the self-
condensation of 3, it was tested whether this mutant also has enhanced aldolase activity towards
the self-condensation of aldehydes 6 and 9. Accordingly, in separate experiments, the purified
enzymes wild-type 4-OT, P1A, F50A and M45Y/F50V (150 µM each) were incubated with
either 6 or 9 (50 mM each) and the reactions were monitored by UV spectroscopy. The results
indicate that the self-condensation of 6 or 9 to give 8 or 11 is enzyme-catalyzed, with variant
M45Y/F50V having strongly enhanced activity when compared to wild-type 4-OT or mutant
F50A (Figures 3 and 4). 1H NMR spectroscopic analysis confirmed the formation of both 7 and 8
as products of the enzyme-catalyzed self-condensation of 6 (Figure S3 and S4), and the formation
of 11 as a product of the enzyme-catalyzed self-condensation of 9 (Figure S5 and S6).
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Figure 3. UV spectra recorded after incubation of 6 with A) no enzyme (control sample); B) 4-OT P1A;
C) wild-type 4-OT; D) 4-OT F50A; and E) 4-OT M45Y/F50V. Formation of 8 (λmax = 227 nm) was
monitored in the course of the reactions.
Figure 4. UV spectra recorded after incubation of 9 with A) no enzyme (control sample); B) 4-OT P1A;
C) wild-type 4-OT; D) 4-OT F50A; and E) 4-OT M45Y/F50V. Formation of 11 (λmax = 240 nm) was
monitored in the course of the reactions.
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Next, we compared the ability of mutants F50A and M45Y/F50V to catalyze an aldol cross-
condensation reaction using 3 and 12 as the substrates. Accordingly, in separate experiments, the
mutant proteins (150 µM) were incubated with 3 (50 mM) and 12 (2 mM) and the reactions were
followed by UV spectroscopy. The UV spectra of the reaction mixture incubated with mutant
F50A showed a decrease in absorbance at 250 nm (Figure 5A), indicating the depletion of 12 as
the result of a cross-coupling reaction. Interestingly, the UV spectra of the reaction mixture with
mutant M45Y/F50V showed a negligible decrease in absorbance at 250 nm (Figure 5B). Instead,
an increase in absorbance at 234 nm was observed, corresponding to the formation of 5 as the
1
result of the self-condensation of 3. H NMR spectroscopic analysis of the reaction mixtures
confirmed that 13 and 14 are the main products in the reaction mixture incubated with mutant
F50A (indicative of cross-coupling), whereas 5 was the main product in the reaction mixture
incubated with mutant M45Y/F50V (Figure S7). Hence, compared to the previously constructed
mutant F50A,[12] mutant M45Y/F50V has an altered substrate specificity and prefers the self-
condensation of 3 over the cross-coupling of 3 and 12. This result is fully consistent with the fact
that mutant M45Y/F50V was engineered for enhanced activity towards the self-condensation of
3.
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Figure 5. UV spectra recorded after incubation of 3 and 12 with A) 4-OT F50A; and B) 4-OT
M45Y/F50V.
In conclusion, we demonstrated that the promiscuous enzyme 4-OT can be engineered into
a more efficient aldolase (variant M45Y/F50V) for self-condensations of aliphatic aldehydes by
exploring small libraries in which only two ‘hotspot’ positions (Met-45 and Phe-50) are varied.
Notably, in a recent study, the same residue positions were identified as ‘hotspots’ for improving
4-OT’s aldolase activity for the cross-condensation of aldehydes 6 and 12, yielding a different 4-
OT variant (M45T/F50A) after combinatorial mutagenesis and activity screening.[20] Hence, 4-
OT can be tailored to catalyze a specific aldol reaction. Work is in progress to determine the
crystal structure of 4-OT variant M45Y/F50V in complex with product 5, 8 or 11. Such enzyme-
product complex structures could guide the design of new 4-OT variants that completely lack
dehydration activity and possess further enhanced aldolase activity, enabling the efficient
enzymatic self-coupling of small aliphatic aldehydes. With only a few natural aldolases available
that can catalyze the self-coupling of aldehydes, including 2-deoxyribose-5-phosphate aldolase
(DERA)[18b] and D-fructose-6-phosphate aldolase (FSA),[19b] the engineering of a promiscuous
enzyme may prove to be a useful approach to create new biocatalysts for these challenging
aldolizations.
MATERIALS AND METHODS
Materials
The sources for the buffers, solvents, components of Luria-Bertani (LB) media as well as the
materials, enzymes and reagents exploited in molecular biology procedures are reported
elsewhere.[25]
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General methods
Standard molecular biology techniques were performed based on methods described
elsewhere.[26] Protein was analyzed by polyacrylamide gel electrophoresis (PAGE) using pre-
casted 10% polyacrylamide gels (NuPAGE® Novex® 10% Bis-Tris). Coomassie brilliant blue
was used to stain the gels. Protein concentrations were determined using the Waddell method.[27]
Enzymatic assays were performed either on a V-650 or V-660 spectrophotometer from Jasco
(IJsselstein, The Netherlands) or on a SPECTROstar Omega plate reader (BMG LABTECH,
Isogen Life Science, de Meern, NL). 1H NMR spectra were recorded on a Varian Inova 500 (500
MHz) spectrometer using a pulse sequence for selective presaturation of the water signal.
Chemical shifts for protons are reported in parts per million scale and are referenced to H2O (δ =
4.80).
Expression and purification of wild-type 4-OT and 4-OT mutants
Wild-type 4-OT and 4-OT mutants were produced in E. coli BL21 (DE3) as native proteins
without an affinity tag, using either a pET20b(+) or pJexpress 414 expression vector. The
construction of the expression vectors for wild-type 4-OT and 4-OT mutants P1A and F50A, as
well as the purification procedure for 4-OT, were reported previously.[10,12]
Cell-free extract (CFE) preparation of E. coli cells expressing the single 4-OT mutants
A defined collection of 1040 single mutant 4-OT genes, each cloned individually into a pJexpress
414 vector, was purchased from DNA2.0 (Menlo Park, CA). An aliquot of each pJexpress 414
vector with a unique mutant 4-OT gene was transformed individually into E. coli BL21 (DE3), as
described elsewhere.[21] Each E. coli BL21(DE3) transformant harboring a pJexpress 414 vector
with a unique mutant 4-OT gene was stored at -80˚C until further use. The expression of the
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mutant 4-OT genes in E. coli BL21(DE3), the preparation of CFEs, and the assessment of the
concentrations of the 4-OT mutants are described elsewhere.[21]
Determination of the aldolase activity of the single 4-OT mutants
The produced CFEs were used to determine the aldolase activity of the single 4-OT mutants
using UV-spectroscopy. The UV-spectroscopic measurements were performed in 96-wells micro
titer plates (MTP) (UV-star µclear, Greiner Bio-one). The final reaction mixtures consisted of
CFE (30% v/v) and propanal (3, 40 mM) in 10 mM NaH2PO4 buffer (pH 7.3). The volume of
these reaction mixtures was 100 µl and the MTPs were sealed with UV-transparent plate seals
(VIEWsealTM, Greiner Bio-one) to prevent evaporation. The MTPs were incubated for 16 h at
25°C during which the reaction progress was monitored by UV spectroscopy (220-500 nm).
Formation of product 5 (λmax = 234 nm) was quantified based on the increase in absorbance at
234 nm. To eliminate false positives in which the increase in absorbance was a result of protein
precipitation (in these cases there is an increase in absorbance over the whole range of the UV-
spectrum), reaction mixtures in which there was a high increase in absorbance at 350 nm (ΔA350
>0.5) were assigned as “precipitation’’ (Figure 1). At this wavelength product 5 has no UV-
absorbance. The aldolase activity of wild-type 4-OT was used as a control and single mutants
with a marked improvement in aldolase activity could be identified. Using Microsoft excel 2010,
the data were represented as colors in the data matrix to create a visually interpretable heat map
(Figure 1).
Construction of double and triple mutant libraries
The construction of the double and triple mutant libraries has been described elsewhere.[20]
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UV spectroscopic assay for the self-condensation of propanal
The self-condensation of propanal (3) was monitored by following the increase in absorbance at
234 nm, which corresponds to the formation of 2-methyl-2-pentenal (5). The enzyme (150 μM)
was incubated in a 1 mm cuvette with 3 (50 mM) in 20 mM NaH2PO4 buffer (pH 7.3; 0.3 mL
final volume), and the reaction was followed for 20 h at room temperature. UV spectra were
recorded from 200 to 400 nm (Figure S1 and Figure 2).
Redissolving WT 4-OT and 4-OT mutants in NaD2PO4 buffer
A VIVASPIN concentrator (from Sartorius Stedim Goettingen, Germany) with a cut-off filter of
5000 Da was washed four times with H2O by centrifugation (4000 rpm, 20 min). Subsequently,
the concentrator was charged with a solution of 4-OT (either WT or mutant; 300 µL with a
concentration of ~10 mg/mL in 20 mM NaH2PO4 buffer, pH 7.3) and centrifuged (4000 rpm, 30
min). The enzyme was retained on the filter and redissolved in NaD2PO4 (200 µL, 20 mM; pD
7.6) and centrifuged (4000 rpm, 30 min). Once more, the remaining enzyme on the filter was
redissolved in NaD2PO4 (300 µL, 20 mM; pD 7.6) after which the final enzyme concentration
was determined. The exchange of NaH2PO4 with NaD2PO4 buffer was carried out only for the
1
enzyme preparations used in the H NMR spectroscopic assay for the self-condensation of 3 or
cross-coupling of 3 and 12.
1H NMR spectroscopic assay for the self-condensation of propanal
4-OT wild-type or mutant (290 µM) was incubated with 3 (30 mM) at room temperature in
NaD2PO4 buffer (20 mM; pD 7.6, final volume of 650 µL in an NMR tube), containing 18-
crown-6 ether as internal standard (2.15 mM). A control sample was prepared containing all
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1
components except for the enzyme. H NMR spectra were recorded ~1 h after the start of the
incubation, and subsequently after 1, 4, 8 and 14 days.
1
Compound 3: H NMR (500 MHz, 20 mM NaD2PO4; pD 7.6): δ 9.69 (t, J = 1.3 Hz, 1H), 2.57
(dq, J = 7.3, 1.3 Hz, 2H), 1.06 (t, J = 7.3 Hz, 3H).
Hydrated form of 3: 1H NMR (500 MHz, 20 mM NaD2PO4; pD 7.6): δ 4.96 (t, J = 5.5 Hz, 1H),
1.59 (dq, J = 7.5, 5.5 Hz, 2H), 0.92 (t, J = 7.5 Hz, 3H).
Compound 5: 1H NMR (500 MHz, 20 mM NaD2PO4; pD 7.6): δ 9.29 (s, 1H), 6.81 (t, J = 7.6 Hz, 1H),
2.41 (dq, J = 7.6, 7.6 Hz, 2H), 1.71 (s, 3H), 1.09 (t, J = 7.6 Hz, 3H).
Internal standard 18-crown-6 (1,4,7,10,13,16-hexaoxacyclooctadecane): 1H NMR (500 MHz,
20 mM NaD2PO4; pD 7.6): δ = 3.68 (s, 24H).
UV spectroscopic assay for the self-condensation of 6
The self-condensation of 6 was monitored by following the increase in absorbance at 227 nm,
which corresponds to the formation of 8. The enzyme (150 µM) was incubated in a 1 mm cuvette
with 6 (50 mM) in 20 mM NaH2PO4 buffer (pH 7.3; 0.3 mL final volume), and the reaction was
followed for 20 h at room temperature. UV spectra were recorded from 200-400 nm (Figure 3).
1H NMR spectroscopic assay for the self-condensation of 6
In separate experiments, 4-OT wild-type or mutant (290 µM, in 20 mM NaH2PO4) was incubated
with 6 (50 mM) at room temperature in NaD2PO4 buffer (20 mM; pD 7.6, final volume of 650 μL
in an NMR tube), containing 18-crown-6 ether as internal standard (2.15 mM). A control sample
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1
was prepared containing all components except for the enzyme. H NMR spectra were recorded
~2 h after the start of the incubation and then after 1, 4 and 7 days.
Compound 6: 1H NMR (500 MHz, 20 mM NaD2PO4; pD 7.6): δ 9.66 (q, J = 3.0 Hz, 1H), 2.23
(d, J = 3.0 Hz, 3H).
Hydrated form of 6: 1H NMR (500 MHz, 20 mM NaD2PO4; pD 7.6): δ 5.23 (q, J = 5.3 Hz, 1H),
1.32 (d, J = 5.3 Hz, 3H).
1
Compound 7: H NMR (500 MHz, 20 mM NaD2PO4; pD 7.6): δ 9.69 (t, J = 5.9 Hz, 1H), 5.16
(m, 1H), 2.03 (m, 2H), 1.18 (d, J = 5.9 Hz, 3H).
1
Compound 8: H NMR (500 MHz, 20 mM NaD2PO4; pD 7.6): δ 9.37 (d, J = 8.3 Hz, 1H), 7.19
(m, 1H), 6.21 (m, 1H), 2.05 (d, J = 7.0 Hz, 3H).
UV spectroscopic assay for the self-condensation of 9
The self-condensation of 9 was monitored by following the increase in absorbance at 240 nm,
which corresponds to the formation of 11. The enzyme (150 µM) was incubated in a 1 mm
cuvette with 9 (50 mM) in 20 mM NaH2PO4 buffer (pH 7.3; 0.3 mL final volume), and the
reaction was followed for 20 h at room temperature. UV spectra were recorded from 200-400 nm
(Figure 4).
1H NMR spectroscopic assay for the self-condensation of 9
In separate experiments, 4-OT wild-type or mutant (290 µM, in 20 mM NaH2PO4) was incubated
with 9 (50 mM) at room temperature in NaD2PO4 buffer (20 mM; pD 7.6, final volume of 650 μL
in an NMR tube), containing 18-crown-6 ether as internal standard (2.15 mM). A control sample
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1
was prepared containing all components except for the enzyme. H NMR spectra were recorded
~2 h after the start of the incubation and then after 1, 4 and 7 days.
1
Compound 9: H NMR (500 MHz, 20 mM NaD2PO4; pD 7.6): δ 9.65-9.67 (m, 1H), 2.50 (m,
2H), 1.59-1.67 (m, 2H), 0.88-0.94 (m, 3H).
Hydrated form of 9: 1H NMR (500 MHz, 20 mM NaD2PO4; pD 7.6): δ 5.03 (t, J = 5.5 Hz, 1H),
1.31-1.43 (m, 2H), 1.53-1.59 (m, 2H), 0.88-0.94 (m, 3H).
1
1
Compound 11: H NMR (500 MHz, 20 mM NaD2PO4; pD 7.6): H NMR (500 MHz, 20 mM
NaD2PO4; pD 7.6): δ 9.26 (s, 1 H), 6.75 (t, J = 7.5 Hz, 1H), 2.42 (q, J = 7.3 Hz, 2H), 2.25 (m,
2H), 1.55 (m, 2H), 0.90-0.97 (m, 3H), 0.90-0.97 (m, 3H)
UV spectroscopic assay for the cross-coupling of 3 and 12
The cross-coupling of 3 and 12 was monitored by following the decrease in absorbance at 250
nm (λmax,12 = 250 nm), which corresponds to the depletion of 12. Simultaneously, the increase in
absorbance at 234 nm, which corresponds to the formation of 5, was monitored. The enzyme
(150 μM) was incubated in a 1 mm cuvette with 3 (50 mM) and 12 (2 mM) in 20 mM NaH2PO4
buffer (pH 7.3; 0.3 mL final volume) and the reaction was followed for 20 h at room temperature.
UV spectra were recorded from 200 to 400 nm.
1H NMR spectroscopic assay for the cross-coupling of 3 and 12
In separate experiments, 4-OT wild-type or mutant (290 µM) was incubated with 3 (30 mM) and
6 (15 mM) in NaD2PO4 buffer (20 mM; pD 7.6, final volume of 650 µL in an NMR tube),
containing 18-crown-6 ether as internal standard (2.15 mM). A control sample was prepared with
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10.1002/cbic.201700121
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1
all the components except for the enzyme. H NMR spectra were recorded ~2 h after the start of
the incubation, and then after 1, 4, 8 and 14 days.
Compound 12: 1H NMR (500 MHz, 20 mM NaD2PO4; pD 7.6): δ 9.94 (s, 1H), 7.97 (d, J = 7.9 Hz, 2H),
7.76 (d, J = 7.5 Hz, 1H), 7.63 (dd, J = 7.9, 7.5 Hz, 2H).
1
Compound 13: H NMR (500 MHz, 20 mM NaD2PO4; pD 7.6): δ 9.74 (s, 1H), 7.48 – 7.36 (m, 5H),
5.23 (s, 1H), 1.03 (s, 3H).
1
Hydrated form of 13: H NMR (500 MHz, 20 mM NaD2PO4; pD 7.6): δ 7.48 – 7.36 (m, 5H), 5.21 (s,
1H), 4.94 (s, 1H), 0.94 (s, 3H).
Compound 14: 1H NMR (500 MHz, 20 mM NaD2PO4; pD 7.6): δ 9.49 (s, 1H), 7.67 (d, J = 7.5 Hz, 2H),
7.55 – 7.50 (m, 4H), 2.03 (s, 3H).
ACKNOWLEDGEMENTS
The authors thank Bimal Prajapati and Thea van den Bosch for their assistance in
characterization of the 4-OT variants. This research was financially supported by the European
Research Council under the European Community's Seventh Framework Programme (FP7/2007-
2013)/ERC Grant agreement n° 242293 (to G.J.P.).
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Table of Contents
Engineering a promiscuous enzyme: we have used a systematic mutagenesis strategy, followed
by combinatorial mutagenesis of identified ‘hotspot’ positions and activity screening, to greatly
enhance the aldolase activity of 4-oxalocrotonate tautomerase (4-OT) for the self-condensation of
linear aliphatic aldehydes yielding α,β-unsaturated aldehydes.
21
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