Characterization of the aldol condensation activity of the trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (tHBP-HA) cloned from Pseudomonas fluorescens N3

Article abstract of DOI:10.1016/j.bbapap.2011.03.013

The gene encoding trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (tHBP-HA) was isolated from Pseudomonas fluorescens N3, an environmental strain able to degrade naphthalene. This enzyme is an aldolase of class I that reversibly catalyzes the transf

Full text of DOI:10.1016/j.bbapap.2011.03.013

Biochimica et Biophysica Acta 1814 (2011) 622–629
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Characterization of the aldol condensation activity of the
trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (tHBP-HA) cloned from
Pseudomonas fluorescens N3
,2
Silvia Ferrara a,1, Erika Mapelli a, Guido Sello b, Patrizia Di Gennaro a,
⁎
a
Department of Environmental Sciences, University of Milano-Bicocca, Piazza della Scienza 1, 20126 Milano, Italy
b
Department of Organic and Industrial Chemistry, University of Milano, Via Venezian 21, 20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 July 2010
Received in revised form 1 March 2011
Accepted 21 March 2011
Available online 7 April 2011
The gene encoding trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (tHBP-HA) was isolated from
Pseudomonas fluorescens N3, an environmental strain able to degrade naphthalene. This enzyme is an aldolase
of class I that reversibly catalyzes the transformation of the trans-o-hydroxybenzylidenepyruvate (t-HBP),
releasing pyruvate and salicylaldehyde. The enzyme was expressed in Escherichia coli as a recombinant
protein of 38 kDa with a His6-Tag at its N-terminus. The recombinant protein His-tHBP-HA was purified by
affinity chromatography and we present here the biochemical characterization of its activity in the aldol
condensation reaction. The aldol condensation reaction parameters were determined using as acceptors both
salicylaldehyde, which is the natural substrate taking part to the naphthalene degradative pathway, and
benzaldehyde. In both cases, His-tHBP-HA shows similar apparent Km and apparent Vmax values. Further
analyses showed that the optimal pH and temperature of His-tHBP-HA activity are 7.0 and 30 °C, respectively.
The tHBP-HA catalytic rates and the availability of an efficient system to produce large amounts of purified
protein are relevant from a biotechnological point of view.
Keywords:
Recombinant production
tHBP-HA
Aldol condensation
Pseudomonas
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
classified according to the donor used [2,3]. The only exception reported
in literature is the novel D-fructose-6-phosphate aldolase isoenzyme
Aldolases are a specific group of lyases that catalyze the reversible
stereoselective addition of a donor compound (nucleophile) to an
acceptor compound (electrophile). Aldolases are known to participate
to both the anabolism and the catabolism of highly oxygenated
metabolites, essential for manybiosynthetic pathways of carbohydrates,
keto acids, and amino acids [1–3]. On the basis of their enzymatic
mechanism, aldolases were grouped into two classes, I and II[2,3]. Class I
aldolases activate the donor substrate by forming a Schiff base as an
intermediate, thanks to a well-conserved lysine in the active site. This
activated donor is then stereoselectively added to the acceptor
aldehyde. In class II aldolases, a divalent metal ion (mostly Zn2+ but
also Co2+ or Fe2+) is coordinated by three histidine residues to the
enzyme active site. The metal cation acts as a Lewis acid and activates
the carbonyl donor substrate. Moreover, aldolases show strict selectivity
for donor compounds, and therefore, they can also be functionally
(FSA) from Escherichia coli as reported by Clapés et al. [3]. This enzyme
which accepts unphosphorylated dihydroxyacetone (DHA) as donor
also readily accepts monohydroxylated donors in place of DHA and
glycolaldehyde (GA) as an alternative donor substrates.
The making and breaking of carbon―carbon (C―C) bonds is a
significant component of synthetic organic chemistry in the formation
of larger and more complex compounds from small and simple
starting materials [4,5]. In addition, C―C bond formation by aldolases
can generate up to two new stereocenters in the resulting aldol
products. Along with the ability to use a wide range of aldehydes as
acceptors, this capacity indicates that most aldolases are interesting
tools in the asymmetric syntheses of rare, deoxy and fluoro sugars,
or sugar-derived compounds as iminocyclitols, statins, epothilones,
D- and L-sialic acids and other analogs [1–3].
The involvement of aldolases in the metabolism of sugars, keto acids,
and amino acids has been extensively described. Examples are
dihydroxyacetone phosphate-dependent aldolases, such as the well-
known fructose 1,6-bisphosphate aldolase [3,4]; aldolases dependingon
pyruvate/phosphoenolpyruvate or acetaldehyde as donor substrates;
and glycine-dependent aldolases, for the formation of hydroxylated
amino acids such as D- and L-threonine and serine [1–3,6].
⁎
Corresponding author at: University of Milano-Bicocca, Piazza della Scienza 1,
20126 Milano, Italy. Tel.: +39 0264482949; fax: +39 0264482996.
E-mail address: patrizia.digennaro@unimib.it (P. Di Gennaro).
Present address: Department of Biomolecular Sciences and Biotechnology,
1
University of Milano, Via Celoria 26, 20133 Milano, Italy.
2
To a lesser extent, aldolase activities were also shown to participate
with their retro-aldol activity in the aromatic hydrocarbon degrading
Present address: Department of Biotechnology and Biosciences, University of
Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.
1570-9639/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbapap.2011.03.013

S. Ferrara et al. / Biochimica et Biophysica Acta 1814 (2011) 622–629
623
Table 1
Strains and plasmids.
pathways in bacteria [7–11]. In particular, Eaton et al. cloned the gene
encoding the trans-o-hydroxybenzylidenepyruvate hydratase-aldolase
(tHBP-HA) from the naphthalene catabolic plasmid NAH7 of Pseudo-
monas putida and showed that this enzyme transforms t-HBP to
salicylaldehyde and pyruvate (Fig. 1) [7]. In addition, the capability of
tHBP-HA to condense aromatic and non-aromatic aldehydes with
pyruvate was assessed usingcrude extracts of recombinantE. coli strains
[8]. Kuhm et al. purified from Pseudomonas vesicularis DSM 6383 (strain
BN6) a trans-o-hydroxybenzylidenepyruvate aldolase [9] which cata-
lyzes the cleavage of trans-o-hydroxybenzylidenepyruvate to salicylal-
dehyde and pyruvate. This reaction is part of the degradative pathways
for naphthalene and naphthalene sulfonate metabolism by P. vesicularis.
In this paper, the purification by conventional methods and the char-
acterization in the retro-aldol activity is reported. Ohmoto described the
presence and the purification of two tHBP-HAs in Sphingomonas
paucimobilis TA-2 [10] involved in naphthalene sulfonate metabolism,
comparing the different characteristics of the tHBP-HA A and the tHBP-
HA B after purification from a cell-free extract.
However, the application of bacterial tHBP-HAs as aldol condensa-
tion tools has not been extensively explored yet; in fact, no previous
studies resulted in the detailed biochemical characterization of the aldol
condensation activity. To address this issue, we aimed at the cloning and
the expression inE. coli of the tHBP-HA from Pseudomonasfluorescens N3
able to degrade naphthalene [12]. The tHBP-HA as a recombinant His-
Tag protein was prepared, purified by affinity chromatography, and we
present here for the first time the biochemical characterization of its
aldol condensation activity using as acceptors both salicylaldehyde,
which is the natural substrate taking part to the naphthalene
degradative pathway, and benzaldehyde.
Relevant genotype and phenotype
Nah+; Sal+
Reference
Strains
Pseudomonas
fluorescens N3
Di Gennaro et al.,
1997
Yanish-Perron
et al., 1985
Escherichia coli JM 109 [recA1 endA1 gyrA96 thi hsdR17
(r+k M+k) supE44 relA1 λ−
Δ(lac-proAB)(F′ traD36 proAB+
lacIq lacZΔM15)]
E. coli M15 [pREP4]
(mal, strr, rifr, thi−, lac−, ara+, gal+
,
Qiagen
mtl−, F−, recA+, uvr+, lon+
)
Plasmids
pVLT33
Kmr, RSF1010-lacIq/Ptac expression
vector, containing MSC of pUC18
Kmr; a pVLT33 derivative containing
the tHBP-HA gene from P. fluorescens
N3 cloned with EcoRI/XbaI ends
de Lorenzo et al.,
1990
This study
pVLT33-ALD
pQE30
Ampr,T5 promoter, lac operator,
ribosome-binding site, start codon,
6×His tag sequence, multiple cloning
site, stop codons in all three reading
frames, Col E1 origin of replication,
lacIq repressor gene.
Qiagen
pQE30-ALD
Ampr, a pQE30 derivative for the
expression of tHBP-HA gene from
P. fluorescens N3 as His-tagged
protein cloned with BamHI/HindIII
This study
that included the gene encoding tHBP-HA (nahE) with its flanking
regions. Forward primer F2 (5′-CAATCGAATTCGGGTGTTTCCATGTC-
GAATAA-3′, EcoRI site underlined) and reverse primer R2 (5′-
CCAACTCTAGAGGAGGTGAACTACTTCAATTCATTA-3′, XbaI site under-
lined) were used to generate an EcoRI/XbaI 1.0 kb DNA fragment
harboring only the nahE gene to be cloned into the vector pVLT33 [15],
givingthe pVLT33-ALD construct. The construct was transferred in E. coli
JM109 by electroporation. pQE30-ALD was generated for tHBP-HA
expression as an N-terminal His6-tagged protein. Direct primer F3 (5′-
CAATCGGATCCATGTCGAATAAAATTATGAA-3′, BamHI site underlined)
and reverse primer R3 (5′-CCAACAAGCTTGGTGAACTACTTCAATTCAT-
TACTG-3′, HindIII site underlined) were used for amplification from
pVLT33-ALD. The pQE30 plasmid (Qiagen, Hilden, Germany) was used
as the expression vector and E. coli M15 (pREP4) as the host strain. All
cloned inserts were verified before use by automated sequencing
(Eurofins MWG, Ebersberg, Germany). The nucleotide sequence of nahE
gene of P. fluorescens N3 is reported in the GenBank DataBase (Accession
number GU319975).
2. Materials and methods
2.1. Bacterial strains and plasmids
The bacterial strains and plasmids used in this paper are listed in
Table 1. P. fluorescens N3 was isolated from activated sludges from a
waste–water treatment plant because of its ability to use naphthalene
as the only carbon and energy source for its growth [12]. E. coli JM109
[13] and E. coli M15 (pREP4) (Qiagen, Hilden, Germany) were chosen
as the host strains: the former was used for gene cloning and
expression, and the latter was well suited for protein purification.
Recombinant plasmids (see below for details) were constructed by
standard procedures [14].
2.2. Identification, PCR-amplification, and cloning of gene encoding
tHBP-HA
2.3. Media, cultivation, and induction conditions
P. fluorescens N3 was grown in M9 mineral medium [16] under a
saturated atmosphere of naphthalene. E. coli recombinant strains were
grown in Luria–Bertani medium or M9 medium [16] supplemented
with 10 mM glucose at 37 °C. Ampicillin and kanamycin were used in
selective media at concentrations of 200 and 100 μg/ml, respectively.
IPTG was added when the culture reached OD600 equal to 0.6, using a
final concentration of 1 mM, and the cultivation temperature was
decreased to 30 °C to minimize the potential formation of inclusion
bodies.
Isolation of the complete coding region of tHBP-HA from
P. fluorescens N3 was obtained from purified total DNA of this strain
by PCR-amplification. The reaction was carried out in 20 μl of a mixture
containing 100 ng of total DNA, 1 μM of each primer, 0.2 mM of dNTPs,
1 U of Pfu DNA polymerase, and PCR buffer (Fermentas, M-Medical,
Milano, Italy). The PCR-amplification was conducted at 95 °C for 30 s,
61.2 °C for 45 s, and 72 °C for 2 min for a total of 35 cycles. Forward
primer F1 (5′-ATCGGACGCCAATTCTGATG-3′) and reverse primer R1
(5′-GGCGCAAGAGATGAGCTTTA-3′) were used to amplify a fragment
OH
OH
2.4. SDS–PAGE analysis of cell extracts
O
O
+
H2O
+
OH
OH
Protein cell extracts from 2 ml of induced cultures were prepared
according to standard procedures [14] and analyzed on NuPAGE
Novex 10% Bis–Tris Gel (Invitrogen, San Diego, CA, USA) using
NuPAGE MOPS SDS as running buffer (Invitrogen, San Diego, CA, USA)
and stained with Coomassie Brilliant Blue R-250. The extracts were
centrifuged to separate the soluble fraction from the inclusion bodies.
The inclusion bodies were analyzed as follows: the pellet containing
CHO
tHBP-HA
O
1
2
3
O
Fig. 1. Reaction catalyzed by the trans-o-hydroxybenzylidenepyruvate hydratase-aldolase
(tHBP-HA) in naphthalene degradative pathway in bacteria belonging to the Pseudomonas
genus. The trans-o-hydroxybenzylidenepyruvate (1) is converted to salicylaldehyde
(2) and pyruvate (3). Salicylaldehyde is a key intermediate that is transformed to catechol,
cleaved by a 2,3-catechol dioxygenase, and then enters in the TCA cycle.

624
S. Ferrara et al. / Biochimica et Biophysica Acta 1814 (2011) 622–629
inclusion bodies was resuspended in BC300 buffer (KCl, 300 mM; Tris,
20 mM pH 7.8; glycerol, 10%) and sonicated 4 times in ice in a
Soniprep 150-MSE (Sanyo MSE, London, UK) with a pulse of 30 s and
2-min intervals between each pulse. The samples were centrifuged for
1 h at 24,000g at 4 °C and the pellets were resuspended in 100 μl 7 M
urea. The samples were sonicated again 4 times with pulses of 10 s
and intervals of 50 s in ice.
The activity was calculated based on the production of the aldol
condensation products and the aldehyde consumption. The specific
activity was defined as the amount of enzyme required to catalyze the
release of 1 μmol of the product per minute under the above
mentioned conditions.
The kinetic parameters of apparent Km and apparent Vmax were
calculated from Lineweaver–Burk plots.
Three independent data sets from the aldol condensation assays
performed with different substrate concentrations were analyzed.
2.5. Western blotting experiments
2.8. His-tagged protein purification
Protein cell extracts were loaded on NuPAGE Novex 10% Bis–Tris Gel
(Invitrogen, San Diego, CA, USA), and after electrophoresis, the proteins
were transferred onto a nitrocellulose membrane (pore size, 0.2 μm;
Amersham Biosciences, Uppsala, Sweden) at 30 V for 1 h in NuPAGE
Novex Tris–Glycine transfer buffer (Invitrogen, San Diego, CA, USA)
using anXCell-Blot Module (Invitrogen, San Diego, CA, USA) for Western
blot analysis. The membrane was blocked with BSA (25 mg/ml) in TBS-T
(TBS with 0.1% Tween 20) at room temperature for 1 h; it was washed 3
times for 10 min with TBS-T and incubated with HisProbe-HRP (Thermo
Scientific Pierce, MA, USA) diluted 1:5000 in TBS-T for 1 h. The mem-
brane was then washed 3 times for 10 min with TBS-T, incubated
with ECL Western Blotting Substrate (Thermo Scientific Pierce, MA,
USA) according to the manufacturer's instructions and visualized by
chemiluminescence.
The purification procedure of His-tHBP-HA protein was performed
using pellet from 500 ml of induced culture broths treated as
described previously. Forty-eight milligrams of crude cell extract of
E. coli M15 (pREP4) (pQE30-ALD) were applied to 2 ml of Co2+ TALON
Metal Affinity Resin (Clontech, Euroclone, Milano, Italy) bed volume.
Washing procedures were carried out according to the manufacturer's
instructions. Proteins were eluted with a linear gradient of imidazole,
from 50 to 250 mM. Eighteen fractions, each one of 1 ml, were
collected and analyzed for their protein content by Bradford assay
(Sigma, St. Louis, MA, USA), SDS–PAGE, and Western blot (50 μg of
sample were loaded per lane) [14]. Imidazole was removed by
membrane dialysis tubes with a cutoff of 12,000 Da against 50 mM
potassium–sodium phosphate buffer (pH 7) overnight at 4 °C.
Fractions were collected and frozen at −20 °C or lyophilized, using
a Bench Lyophilizator LIO-5P (Cinquepascal S.r.l., Milano, Italy). The
lyophilized protein was stored at −20 °C.
2.6. Preparation of cell extracts for aldol condensation assays
Cells from 500 ml of induced cultures were collected by centrifuga-
tion at 4 °C, washed twice, and resuspended in 50 mM potassium–
sodium phosphate buffer (pH 7); the suspension was sonicated 4 times
in ice in a Soniprep 150-MSE with a pulse of 30 s and 2-min intervals
between each pulse. The lysate was cleared for 1 h at 24,000g.
2.9. Characterization of transformation products
The aldol condensation reactions were followed by HPLC anal-
yses using a Waters 515 HPLC instrument (Waters, Milford, MA, USA)
coupled with a Waters 2487 Dual λ Absorbance Detector and a
Millennium32 Waters data analysis program. The column was an Alltech
Adsorbosphere XL C18 5U (length, 250 mm; ID, 46 mm) (Alltech,
Bologna, Italy). Flow rate was set at 0.8 ml/min using water:acetonitrile
1:1 as the mobile phase. The double-beam detector allowed the visu-
alization of substrate consumption salicylaldehyde or benzaldehyde at
256 nm and product formation trans-o-hydroxybenzylidenepyruvate or
trans-benzylidenepyruvate at 296 nm.
The isolation and identification of the condensation products was
performed as follows. For benzaldehyde, the reaction mixture (10–
100 ml) was acidified to pH 3.0 and the products were extracted 3
times with an equal volume of ethyl acetate; the collected organic
phases were dried over Na2SO4 and the solvent was removed in
vacuum; the residue was dissolved in CDCl3. For salicylaldehyde, the
reaction mixture was frozen at −40 °C, lyophilized for 24 h, and the
residue directly extracted with DMSO-d6. 1H-NMR and 13C-NMR
analyses were performed using a Brucker AC-200 NMR.
2.7. Enzymatic assays for aldol condensation reaction
Aldol condensation assays using crude extracts were performed at
30 °C in 50 mM potassium–sodium phosphate buffer (pH 7) with 0.2–
1.6 mg of total proteins, 10 mM of pyruvate (donor), and 0.1 mM of
salicylaldehyde or benzaldehyde (acceptor), or 1 M of pyruvate (donor)
and 10 mM of salicylaldehyde or benzaldehyde (acceptor). The re-
actions were performed in a final volume of 2 ml and followed for 3 h.
Aldol condensation assays for kinetics parameter determination
using purified His-tHBP-HA were performed using 0.001–0.01 mg/ml
of purified protein, 0.08–2 M of donor compound, and 0.8–20 mM of
acceptor compound in a final volume of 2 ml in the same conditions
utilized for the crude extracts.
Preparative reactions with crude extract were carried out at 30 °C in
a final volume of 100 ml using 100 mg of total proteins dissolved in
50 mM potassium–sodium phosphate buffer (pH 7), and 20 mM of
donor and 1 mM of acceptor substrates. The reactions were stopped
after 7 h.
2.10. Chemicals
In case of His-tHBP-HA, preparative reactions were carried out at
30 °C in a final volume of 10 ml using 0.1–1 mg/ml of purified protein
dissolved in 50 mM potassium–sodium phosphate buffer (pH 7), and
20 mM of donor and 1 mM of acceptor substrates. The reactions were
stopped after 3 h.
Acetic acid, benzaldehyde, ethylacetate, naphthalene, isopropyl-ß-
D-thiogalactopyranoside (IPTG), sodium pyruvate, salicylaldehyde,
and sodium acetate were supplied by Sigma-Aldrich.
Temperature activity assays were performed in 50 mM potassi-
um–sodium phosphate buffer (pH 7) with 0.01 mg/ml of purified
protein, 10 mM benzaldehyde, and 1 M pyruvate in a final volume of
2 ml, at T from 25 to 40 °C.
3. Results
3.1. Identification and cloning of the gene encoding tHBP-HA from
P. fluorescens N3
pH activity assays were performed at 30 °C using 0.01 mg/ml of
purified protein, 10 mM benzaldehyde, and 1 M pyruvate in a final
volume of 2 ml. The following buffers were used: 50 mM sodium
acetate (pH 5.5–6.5); 50 mM potassium–sodium phosphate (pH 6.5–
7.5); 50 mM Tris–HCl (pH 7.5–8.5).
In order to isolate a gene encoding a trans-o-hydroxybenzylide-
nepyruvate hydratase-aldolase (tHBP-HA) activity from P. fluorescens
N3, putative flanking sequences needed to be initially identified. To
this end, the amino acid sequence (Swiss-Prot entry Q51947) of tHBP-

S. Ferrara et al. / Biochimica et Biophysica Acta 1814 (2011) 622–629
625
HA encoded by the nahE gene carried by the NAH7 plasmid of P. putida
G7 was used as bait in reiterated BLASTp [17] searches. This analysis
resulted in a set of proteins with a specific degree of similarity to the
NAH7 tHBP-HA.
product was recovered and identified as trans-o-hydroxybenzylide-
nepyruvate by 1H-NMR and 13C-NMR analysis (Table 2).
In an attempt to influence the equilibrium of the reaction in favor
of the aldol condensation product and further characterize the
enzyme behavior, benzaldehyde was used as an acceptor. In fact, in
the literature [8], it is reported that the trans-benzylidenepyruvate
retro-aldol reaction is 200 times slower than that of trans-o-
hydroxybenzylidenepyruvate. The reaction was followed by HPLC
analysis and a decreasing peak corresponding to benzaldehyde
consumption was detected at 256 nm together with a growing peak
at 296 nm that can be assigned to product formation (Fig. 2B). The
product was extracted for 1H-NMR and 13C-NMR analysis after a
preparative reaction, resulting in the trans-benzylidenepyruvate (t-
BP) (Table 2).
The DNA sequences upstream and downstream of the genes
encoding this set of proteins were aligned with ClustalW [18] to
detect conserved anchor sequences (data not shown). Several
forward and reverse PCR primers were designed within conserved
flanking sequences. Different pairs of these primers were used in PCR
reactions with the P. fluorescens N3 total DNA as template. A primer
pair composed of oligos F1 and R1, annealing to the upstream and the
downstream regions, amplified a 1.1 kb fragment that was then
sequenced and found to include a 1.0 kb ORF sharing 94% of sequence
identity with the NAH7 nahE gene of the G7 strain. In order to amplify
the N3 nahE gene alone with no adjoining intergenic sequences,
primers F2 and R2 were designed. Finally, a construct was generated
by cloning the 1.0 kb PCR fragment amplified from P. fluorescens N3
into the broad host range lacIq/Ptac-based vector pVLT33 [15], giving
rise to the construct pVLT33-ALD. E. coli JM109 was chosen as host
strain.
3.3. Expression and purification of the His-tHBP-HA protein
In order to characterize the biochemical and kinetic parameters of
the aldol condensation reaction, the tHBP-HA protein was engineered
at its N-terminus with a His6-Tag and expressed in E. coli M15
(pREP4) carrying pQE30-ALD.
3.2. Expression of the tHBP-HA in E. coli and aldol condensation assay in
crude extracts
To assess the capability of E. coli M15 (pREP4) host strain to
efficiently express the His-tagged tHBP-HA of P. fluorescens N3,
samples of induced culture were taken after 4, 6, and 24 h from the
inducer addition, and the protein content of the soluble fraction and
inclusion bodies was analyzed by Bradford assay and SDS–PAGE (data
not shown). In the soluble fraction, a band corresponding to the
expected molecular weight (38 kDa) increased and a maximum level
of expression was observed 6 h after induction (Fig. 3, lane 2).
Western blot analysis with HisProbe-HRP performed on these same
samples revealed a unique band of 38 kDa corresponding to the one
visualized by SDS–PAGE; this further indicated that the His-tagged
aldolase had been expressed.
The tHBP-HA from P. fluorescens N3 was expressed in E. coli JM109
(pVLT33-ALD) strain and enzyme capability to perform aldol conden-
sation of salicylaldehyde and pyruvate was tested using crude cell
lysates.
The aldol condensation reaction experiment was followed by HPLC
analysis monitoring the substrate consumption (salicylaldehyde) and
product formation (trans-o-hydroxybenzylidenepyruvate) (Fig. 2A).
In order to ensure that the product of the aldol condensation was
the trans-o-hydroxybenzylidenepyruvate, a preparative reaction was
performed. This reaction was stopped after 7 h, when the peak
corresponding to the concentration of salicylaldehyde appeared to be
almost halved with respect to the initial concentration. The reaction
The aldol condensation activity of crude extract (0.1 mg/ml) of
E. coli M15 (pREP4) carrying pQE30-ALD was tested using pyruvate
(1 M) and benzaldehyde (10 mM) as substrates. HPLC analysis
A
0.25
0.20
0.15
0.10
0.05
0.00
0.25
0.20
0.15
0.10
0.05
0.00
0.25
T0
T30
T60
0.20
0.15
0.10
0.05
0.00
0.0 1.00 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
Min
Min
Min
B
0.5
0.5
0.5
0.4
0.3
0.2
0.1
0.0
T0
T30
T60
0.4
0.3
0.2
0.4
0.3
0.2
0.1
0.0
0.1
0.0
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
Min
Min
Min
Fig. 2. HPLC analysis of aldol condensation of benzaldehyde/salicylaldehyde and pyruvate catalyzed by cell extracts of Escherichia coli JM109 (pVLT33-ALD) strain as described in
Materials and methods. The solid line is the absorbance at 256 nm; the dotted line is the absorbance at 296 nm. The retention time around 3 min is product condensation signal; the
retention time around 7 min is aldehyde signal. Part A, salicylaldehyde reaction; part B: benzaldehyde reaction.

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S. Ferrara et al. / Biochimica et Biophysica Acta 1814 (2011) 622–629
Table 2
3.4. Biochemical characterization and kinetic parameter determination
1H-NMR and 13C-NMR data of the aldol condensation reaction by tHBP-HA of P. fluorescens
N3.
The kinetic characterization of the condensation reaction of the
purified His-tHBP-HA was performed in the presence of pyruvate as
donor and using as acceptors either salicylaldehyde or benzaldehyde,
with 0.001 mg/ml or 0.01 mg/ml of purified protein, respectively.
Pyruvate was added at concentration ranging from 0.08 M to 2 M
(100 times the concentration of acceptors), while the salicylaldehyde or
benzaldehyde concentrations ranged from 0.8 mM to 20 mM. The
reported values are the average of three independent experiments.
Kinetic parameters of the recombinant His-tHBP-HA were determined;
the results are shown in Fig. 4. The values of apparent Km and apparent
Vmax were calculated from Lineweaver–Burk plots. The apparent Km and
apparent Vmax values for benzaldehyde and salicylaldehyde were 8.98
and 3.58 mM, and 17.24 and 35.46 μmol mg−1 min−1, respectively.
For further characterization of the recombinant His-tHBP-HA, the
effects of temperature and pH on enzyme activity with 1 M donor and
10 mM acceptor were studied. At a fixed pH of 7.0, maximum His-
tHBP-HA activity was observed at 40 °C (Fig. 5A). Further temperature
increase caused a decrease of the specific activity. A control of the
enzyme stability at selected temperatures (40 °C, 35 °C, and 30 °C)
was then performed; the enzyme was kept at these temperatures for
30 min and then an enzymatic assay was run. The decreases of the
activity were 64% at 40 °C and 24% at 35 °C, while at 30 °C, the activity
remained unchanged. However, at 30 °C, the decreases of the activity
were 24% after 5 h and 54% after 24 h. Hence, 30 °C was the selected
temperature of the successive enzymatic activity measurements.
The effect of pH on the enzyme activity was studied. Fig. 5B shows
the initial reaction rate measured by the aldol condensation assay
method using benzaldehyde (10 mM) and pyruvate (1 M) as sub-
strates with the following buffers: acetate (pH 5.5–6.5), potassium
phosphate (pH 6.5–7.5), and Tris–HCl (pH 7.5–8.5). The maximum
activity of His-tHBP-HA was at pH 7.0. These data confirm that the
conditions of 30 °C and pH 7.0 are the best choice for running the
enzyme assays.
Compound
1H-NMR and 13C-NMR
CDCl3, δ: 7.53, 1H, d, J=16.0 Hz; 7.6–7.64,
3H, m; 7.86, 2H, dd, J=2.3 Hz, 9 Hz; 8.0,
1H, d, J=16.0 Hz
CDCl3, δ: 196.80 1C (s); 167.5 1C (s);
151.574, 1C (d); 128.721–142.773, 5C
(d)+1C (s); 117.840 1C (d)
O
OH
O
Trans-benzylidenepyruvic acid
DMSO-d6, δ: 6.72, 1H, d, J=16.2 Hz, 6.8–
6.9, 2H, m; 7.2, 1H, d, J=8.1 Hz; 7.52, 1H,
dd, J=8.1, 1.0 Hz; 7.6, 1H, d, J=16.2 Hz
DMSO-d6, δ: 198.4 1C (s); 168.6 1C (s);
158.2 1C (s); 140.5 1C (d); 132.4 1C (d);
129.1 1C (d); 125.2 1C (d); 121.0 1C (s);
120.2 1C (d); 117.4 1C (d)
OH
O
O-
O
Trans-o-hydroxybenzylidenepyruvate
showed that initial amount of benzaldehyde had been completely
consumed in 120 min. These data led to the quantification of the
specific activity of the crude extract corresponding to 4.59 μmol mg−1
min−1
.
His-tHBP-HA was prepared in a soluble form by a metal-affinity
purification procedure. Thewhole soluble fraction, containing 6.85 mgof
total proteins in a final volume of 7 ml, was loaded onto the Co2+ metal-
affinity column. Fractions were pooled and analyzed to determine their
protein content by Bradford assay, SDS–PAGE (Fig. 3, lanes 3–9), and
Western blot analysis. Successively, imidazole was removed by dialysis
and the fractions were stored at −20 °C.
The enzymatic activity of the fractions containing the recombinant
His-tHBP-HA was tested by condensation assays using 1 M pyruvate and
10 mM benzaldehyde as substrates in 2-ml reactions at 30 °C. The initial
amount of benzaldehyde was halved in 30 min, while a peak correspond-
ing to the trans-benzylidenepyruvate increased as the reaction pro-
gressed. From the analysis of the kinetics, the specific activity of the His-
3.5. Enzyme stability of lyophilized His-tHBP-HA
tHBP-HA was established to be 9.56 μmol mg−1 min−1
.
We also tested the level of protein enzymatic activity after
lyophilization. We compared the performance of the purified compo-
nent before and after lyophilization. Approximately 0.02 mg of
lyophilized protein was rehydrated in 50 mM sodium–phosphate
buffer, pH 7.0; then, the aldol condensation activity was assayed vs.
the same amount of protein not lyophilized, using 1 M pyruvate and
10 mM benzaldehyde. No significant difference was detected between
the two reactions.
4. Discussion
It is known that the catabolic pathway of naphthalene metabolism
in Pseudomonas includes a retro-aldol reaction that transforms trans-
o-hydroxybenzylidenepyruvate into pyruvate and salicylaldehyde
(Fig. 1). The reaction is described as being reversible and not needing
any cofactors [7,9,19]. Several aldolases belonging to the pathways of
sugar metabolism have been described in detail [1–3]; in contrast,
limited data are available concerning the aldol activity of aldolases
belonging to other pathways [7,9,11]. In order to contribute some
more data, in this work, we report the cloning of the gene and the
isolation and characterization of the corresponding tHBP-HA enzyme
from P. fluorescens N3.
The amino acidic sequence of the 37 kDa protein from P. fluorescens
N3 shares 95% of similarity with the tHBP-HA of P. putida G7 strain.
Besides, the N3 tHBP-HA enzyme probably belongs to the group that
forms Schiff bases as is the case for the tHBP-HA of P. putida G7 [7–9].
This is supported by the presence in the sequence of the motif GxxGE
involved in the binding of the alpha-keto acid moiety and of the highly
Fig. 3. Purification of His-tHBP-HA performed by a metal-affinity chromatography from
crude extract of Escherichia coli M15 (pREP4) (pQE30-ALD) strain induced with IPTG
1 mM in LB medium at 30 °C for 6 h. SDS–PAGE analysis of some eluted fractions: lane
M, molecular mass markers; lane 1, E. coli JM109 (pVLT33) extract, the negative control;
lane 2, E. coli M15 (pREP4) (pQE30-ALD) extract; lane 3, fraction 3 (imidazole, 50 mM);
lane 4, fraction 4 (imidazole, 50 mM); lane 5, fraction 6 (imidazole, 150 mM); lane 6,
fraction 7 (imidazole, 150 mM); lane 7, fraction 8 (imidazole, 150 mM); lane 8, fraction
11 (imidazole, 250 mM); lane 9, fraction 15 (imidazole, 250 mM).

S. Ferrara et al. / Biochimica et Biophysica Acta 1814 (2011) 622–629
627
A
B
14
12
10
8
40
35
30
25
20
15
10
5
6
4
2
0
0
0
5
10
15
20
25
0
5
10
15
20
25
[SA](mM)
[BA](mM)
y = 0.521x + 0.058
R2= 0.991
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
y = 0.1011x + 0.0282
R2= 0.997
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
-0.5
0
0.5
1
1.5
-0.5
0
0.5
1
1.5
-0.1
[1/BA](1/mM)
[1/SA](1/mM)
Fig. 4. Michaelis–Menten curves and Lineweaver–Burk plots of the His-tHBP-HA aldol condensation reaction with various concentrations of benzaldehyde (A) and salicylaldehyde
(B). Reactions were performed under the aldol condensation assay conditions described in Materials and methods; concentrations of salicylaldehyde and benzaldehyde are indicated
in the figure.
conserved lysine (Lys183) involved in the Schiff base formation
[8,9,20,21].
Other genes encoding the trans-o-hydroxybenzylidenepyruvate
hydratase-aldolase (tHBP-HA) from the naphthalene catabolic pathway
that can transform t-HBP to salicylaldehyde and pyruvate have been
reported [7–9].
In particular, Eaton et al. reported the capability of NAH7 tHBP-HA to
transform different substrates into aromatic and non-aromatic alde-
hydes and pyruvate using crude extracts of recombinant E. coli strains
[8], but no kinetic parameter with the purified enzyme was determined.
Nevertheless, the authors reported the t-HBP rates which varied from
1.6 to 3.6 μmol mg−1 min−1 measured using the crude extract; in
addition, a Km value of 4 μM for t-HBP was reported by Kuhm et al. [9].
Ohmoto et al. reported the purification of two tHBP-HAs, A and B,
from S. paucimobilis TA-2; their Km values for t-HBP were 9 and 3 μM,
respectively [10].
Finally, Kuhm et al. purified from P. vesicularis DSM 6383 (strain
BN6) a trans-o-hydroxybenzylidenepyruvate aldolase [9]. In this
paper, the purification by conventional methods and the character-
ization of the retro-aldol activity is reported. The Km value of 17 μM for
t-HBP in contrast with the value of 0.4 mM for t-BP was determined.
We present here for the first time the characterization of the aldol
condensation activity of a tHBP-HA. This extends and complements the
literature data [8–10] that describe the characterization of the retro-
aldol activity.
In our case, the successful determination of the kinetic parameters in
the condensation reaction was made possible by the availability of an
efficient integrated production process based on the use of an expression
system yielding intracellular level of 50% of the total soluble protein that
allowed the study and characterization of the His-tHBP-HA enzyme.
Since the equilibrium in the reaction of salicylaldehyde favors
cleavage while retro-aldol reaction of benzylidenepyruvate is very slow
A
100
80
60
40
20
0
B
100
80
60
40
20
0
20
30
40
50
60
5
6
7
8
9
temperature (°C)
pH
Fig. 5. Temperature (A) and pH (B) effects on the activity of His-tHBP-HA. Reactions were performed under the aldol condensation assay conditions described in Materials and
methods; temperature range was 25–50 °C, pH range was 5.5–8.5 (pH 5.5–6.5 acetate buffer, dotted line; pH 6.5–7.5 phosphate buffer, solid line; pH 7.5–8.5 Tris–HCl buffer, dotted
line).

628
S. Ferrara et al. / Biochimica et Biophysica Acta 1814 (2011) 622–629
H+
R
R
O
HN
N
OH
NH
R
H C
3
2
+
OH
OH
H C
2
I
O
II
H
O
O
B
H+
H
H
O
H+
OH
+
R
R
O
R
B
N
N
III
IV
+
N
H
OH
OH
OH
H
H C
2
O
O
B
O
Fig. 6. Hypothetical mechanism of the aldol condensation reaction catalyzed by tHBP-HA. Schiff base formation (I); imine–enamine equilibrium (II); aldol condensation reaction
(III); aldol dehydratation (IV).
[8], and because it is known that the tHBP-HA catalyzes the
condensation reaction of several aromatic aldehydes including benzal-
dehyde [8], we used also this molecule as the acceptor for aldol
condensations.
Acknowledgments
The authors are particularly grateful to G. Bertoni and P. Parenti for
helpful discussion and critical reading of the manuscript, and E.
Lanfranchi for technical support. This work was supported by MIUR-
PRIN 2007 (grant 20077MY8M9 004) and was the recipient of a
Sovvenzione Globale Ingenio fellowship granted to Silvia Ferrara from
the Lombardia Regional Government.
The apparent Km values show that His-tHBP-HA has a similar affinity
for benzaldehyde (8.98 mM) and salicylaldehyde (3.58 mM) that is the
natural substrate taking part to the naphthalene degradative pathway.
The apparent Vmax values are also similar: the salicylaldehyde apparent
Vmax (35.46 μmol mg−1 min−1) is two-fold the benzaldehyde apparent
Vmax (17.24 μmol mg−1 min−1). However, considering that cleavage
reaction rate of t-HBP is reported to be 200 times greater [8] than t-BP
rate, it is evident that the measured salicylaldehyde condensation rate is
lower than the actual rate. This result could be explained by the
participation of the acidic phenol proton to the several acid–base
reactions present in the hypothetical mechanism [8] (Fig. 6).
The comparison of the enzyme affinity for substrates and products
and of enzymatic rates is not easy; in fact, all the available literature data
refer to the reaction performed from the unsaturated acids to the
corresponding aldehydes and pyruvate. Nevertheless, we can attempt a
qualitative comparison. Concerningsubstrate affinity, it appears that the
values reported by Kuhm et al. [9] showed a higher affinity with t-HBP
(4–20 μM) than that we determined for salicylaldehyde (3.58 mM); in
contrast, the value that we obtained with benzaldehyde (8.98 mM) is
more similar to the value obtained by Kuhm et al. for the transformation
of the t-BP into benzaldehyde and pyruvate (0.4 mM). Concerning rates,
Eaton et al. [7,8] report rates measured using crude extracts; these were
in the range of 1.6 to 3.6 μmol min−1 mg−1. On the other hand, Kuhm et
al. report a specific rate of 23.7 μmol min−1 mg−1 for the purified
enzyme. Both these values are similar to our calculated rate of 35.5 μmol
min−1 mg−1, and all refer to the reaction of t-HBP and salicylaldehyde.
Our results also show that the optimum pH and temperature of the
enzyme activity are 7.0 and 30 °C, respectively. The aldolase activity
rapidly decreases even at pH 7.5 or 6.5 (−20%); in contrast, it increases
at temperatures between 30 °C and 40 °C (+60%). However, experi-
ments performed to test the enzyme stability show an activity decrease
of 64% at 40 °C and 24% at 35 °C, after 30 min. In addition, at 30 °C, the
activity decrease was 24% after 5 h and 54% after 24 h.
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