Purification and enzymatic characterization of trans-o-hydroxybenzylidenepyruvate hydratase-aldolase from Rhodococcus opacus and enzymatic formation of α, β-unsaturated ketones

Article abstract of DOI:10.1080/09168451.2019.1625262

Trans-o-hydroxybenzylidenepyruvate (tHBPA) hydratase-aldolase (RnoE) catalyzes the conversion of tHBPA to 2-hydroxybenzaldehyde and pyruvate. We purified RnoE from Rhodococcus opacus and characterized its enzymatic properties. It exhibited maximum enzyme activity at 60°C and catalyzed the reverse reaction, converting various aromatic benzaldehydes and pyruvate to benzylidenepyruvate, indicating that this enzyme can be adapted for the enzymatic synthesis of α, β-unsaturated ketones.

Full text of DOI:10.1080/09168451.2019.1625262

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: https://www.tandfonline.com/loi/tbbb20
Purification and enzymatic characterization of
trans-o-hydroxybenzylidenepyruvate hydratase-
aldolase from Rhodococcus opacus and enzymatic
formation of α, β-unsaturated ketones
Toshihiro Suzuki & Noboru Takizawa
To cite this article: Toshihiro Suzuki & Noboru Takizawa (2019): Purification and enzymatic
characterization of trans-o-hydroxybenzylidenepyruvate hydratase-aldolase from Rhodococcus
opacus and enzymatic formation of α, β-unsaturated ketones, Bioscience, Biotechnology, and
Biochemistry, DOI: 10.1080/09168451.2019.1625262
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BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
https://doi.org/10.1080/09168451.2019.1625262
NOTE
Purification and enzymatic characterization of trans-o-
hydroxybenzylidenepyruvate hydratase-aldolase from Rhodococcus
opacus and enzymatic formation of α, β-unsaturated ketones
Toshihiro Suzukia and Noboru Takizawab
b
aDepartment of Fermentation Sciences, Faculty of Applied Biosciences, Tokyo University of Agriculture, Tokyo, Japan; Department of
Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University of Science, Okayama, Japan
ABSTRACT
ARTICLE HISTORY
Received 2 April 2019
Accepted 20 May 2019
Trans-o-hydroxybenzylidenepyruvate (tHBPA) hydratase-aldolase (RnoE) catalyzes the
conversion of tHBPA to 2-hydroxybenzaldehyde and pyruvate. We purified RnoE from
Rhodococcus opacus and characterized its enzymatic properties. It exhibited maximum
enzyme activity at 60°C and catalyzed the reverse reaction, converting various aromatic
benzaldehydes and pyruvate to benzylidenepyruvate, indicating that this enzyme can be
adapted for the enzymatic synthesis of α, β-unsaturated ketones.
KEYWORDS
Rhodococcus; naphthalene
degradation; tHBPA;
hydratase-aldolase; aldol
reaction
Microorganisms degrade polycyclic aromatic
hydrocarbons (PAHs), for example, naphthalene,
phenanthrene, and anthracene, via 4-substituted
2-ketobut-3-enoate (α, β-unsaturated ketones)
intermediates [1]. Among α, β-unsaturated ketones,
trans-o-hydroxybenzylidenepyruvate (tHBPA) is
produced as an intermediate in the upper pathway
of microbial PAH-degradation and is converted to
hydroxyl-aromatic aldehyde and pyruvate by
m-hydroxybenzylidenepyruvate
(mHBPA)
and
trans-p-hydroxybenzylidenepyruvate (pHBPA) were
similarly synthesized using the same reaction.
To obtain native RnoE, the R. opacus strain
CIR2 was cultured for 45 h at 30°C with shaking
in W minimal salt medium [8] containing 0.3%
solid naphthalene (Nacalai Tesque, Kyoto, Japan)
as the sole carbon and energy source. Cells were
collected and washed twice with 50 mM P Buffer
(50 mM Na2HPO4 and 50 mM KH2PO4, pH 7.5)
and then re-suspended in the same buffer (5×the
cell pellet wet-weight). Re-suspended cells were
disrupted using a Multi-Beads-Shocker (Yasui
Kikai, Osaka, Japan), and the lysate was harvested
by ultracentrifugation at 92,300 × g for 60 min at
4°C.
Enzyme activity was measured by monitoring the
decrease in absorbance at 340 nm, the λmax of tHBPA
[4], at 30°C. Enzyme activity was calculated using
a molar extinction coefficient of 12.8 mM−1 for
tHBPA [7], and one unit of enzyme was defined as
the amount of enzyme that produces one μmole of
2-hydroxybenzaldehyde per min at 30°C. Protein
concentration was determined using the method of
Bradford [9], and bovine serum albumin was used as
standard.
hydratase-aldolase (via
a
retro-aldol reaction)
[2,3]. Hydratase-aldolase is widely distributed in
PAH-degrading bacteria and exhibits a unique
activity that catalyzes α, β-unsaturated ketone
hydroxylation and aldol cleavage [4].
tHBPA hydratase-aldolase (RnoE) converts
tHBPA to 2-hydroxybenzaldehyde and pyruvate
(Figure 1(a)). In addition, RnoE has been known to
catalyze the reverse aldol reaction and produce var-
ious α, β-unsaturated ketones from several alde-
hydes and pyruvate [3]. Thus, this enzyme is not
only important for microbial degradation of PAHs
but also suited to being a biocatalyst for the synth-
esis of various useful compounds. However, only
a few reports on its enzymatic characterization and
function have been published [3,5–7]. Here we pur-
ified native RnoE from the naphthalene-degrading
soil bacterium Rhodococcus opacus CIR2 and inves-
tigated its enzymatic properties and aldol reaction
activity using several aromatic aldehydes and
pyruvate.
The cell-free extract reduced the intensity of the
absorbance peaks of 2-hydroxybenzaldehyde at 296
nm and 340 nm and increased absorbance at 255
nm (Figure 1(b)) [4]. In addition, a retro-aldol
reaction with the extract using tHBPA as
a substrate was analyzed by HPLC (LC-10A system;
Shimadzu, Japan) with a Luna 5μ C-18 column
(Ø4.6 mm × 250 mm; Shimadzu). The analysis
Owing to its commercial unavailability, tHBPA
and its derivative were synthesized by an aldol
condensation reaction from 2-hydroxybenzaldehyde
and pyruvate under alkaline conditions. Trans-
CONTACT Toshihiro Suzuki
ts206188@nodai.ac.jp
© 2019 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
T. SUZUKI AND N. TAKIZAWA
a)
b)
2.25
0
250
400
Wavelength (nm)
c)
d)
1
1
(I) No enzyme
M
1
2
3
4
5 M
(kDa)
83.0
62.0
47.5
32.5
25.0
2
2
3
3
16.5
(II) Enzyme added
6.5
0
20
5
10
15
Retention time (min)
Figure 1. Enzymatic reactions and purification of RnoE.
(a) Catalytic activity of RnoE. (b) Spectrophotometric analysis of the enzyme reaction using RnoE and tHBPA with a Shimadzu UV-Vis UV-1200
spectrophotometer. The upward arrow indicates the 255 nm peak, whereas the downward arrows indicate the 296 nm and 340 nm peaks. (c) HPLC
analysis of tHBPA hydrolysis in the presence (I) or absence (II) of RnoE. Peak 1 = tHBPA, peak 2 = 2-hydroxybenzaldehyde and peak 3 = pyruvate. (d)
SDS-PAGE of each step in the purification of RnoE. Lane M, molecular weight markers; lane 1 cell-free extract; lane 2, active fraction from DEAE-Toyoperl
650 M; lane 3, active fraction from HiLoad 16/10 Phenyl Sepharose; lanes 4 and 5, active fractions from MonoQ HR 5/5. Marker sizes (kDa) are indicated
on the left. All proteins were stained with Coomassie brilliant blue R-250.
revealed an increase in 2-hydroxybenzaldehyde and
a decrease in tHBPA levels (Figure 1(c)), indicating
that the strain CIR2 possesses a soluble form of
RnoE.
A summary of the enzymatic characterization of
purified RnoE and a comparison to other tHBPA
hydratase-aldolases is shown in Table 1. The opti-
mum pH and temperature of RnoE were 7.5–8.0 and
60°C, respectively. PahE also exhibited the same
optimum temperature. To date, there have been
few reports on the enzymatic characterization of
hydratase-aldolases using tHBPA as substrate;
those that exist used enzymes from P. vesicularis
DSM6383 [7], P. fluorescens N3 [2], and
Sphingomonas paucimobilis TA-2 [6]. The optimum
pH of RnoE was similar with that of enzymes from
these other known species, although the optimum
reaction temperature of hydratase-aldolases/RnoE
has not been reported for other species except for
P. fluorescens.
The substrate specificity of RnoE was investi-
gated using trans-m-hydroxybenzylidenepyruvate
(mHBPA) and trans-p-hydroxybenzylidenepyruvate
(pHBPA), which were synthesized by chemical
aldol condensation reactions using 3- or 4-hydro-
xybenzaldehyde and pyruvate. RnoE was equally
active using pHBPA as a substrate compared with
tHBPA but only 32% as active when using mHBPA.
Conversely, the activity of PahE with pHBPA
Purification of native RnoE was performed using
DEAE Toyopearl 650 M (Tosoh, Tokyo, Japan),
HiLoad 16/10 Phenyl Sepharose (Amersham), and
MonoQ HR 5/5 (Amersham) connected to an FPLC
system (Amersham, Sweden). Purity at each purifi-
cation step was assessed by SDS-PAGE. The final
material produced a single band of 32 kDa on SDS-
PAGE (Figure 1(d)) and was purified 23.9-fold with
a yield of 0.6% from the lysate. Gel filtration on
Sephadex 200 HR 10/300 (Amersham) gave
a native molecular mass of 64.5 kDa, suggesting
that the enzyme is a homodimeric complex. It has
been reported that the native molecular mass of
Pseudomonas vesicularis DAM6383 is 120 kDa and
that it is consists of a homotrimeric complex [7]. In
addition, to compare native molecular mass and
enzymatic properties, we also purified the native
tHBPA hydratase-aldolase (PahE) from another
naphthalene degrader, Pseudomonas aeruginosa
PaK1 [10], and determined it to be a homotetra-
meric complex.

THBPA HYDRATASE-ALDOLASE FROM RHODOCOCCUS OPACUS
3
and mHBPA relative to tHBPA was 26% and 68%,
respectively.
The Km and Vmax of RnoE for tHBPA were deter-
mined by linear regression using Lineweaver–Burk
plots. The Km value was 20 µM, as was for the enzyme
from P. vesicularis DSM6383, and the Vmax was 0.82
µmol/min/mg. The Km and Vmax values of PahE of
P. aeruginosa Pak1 were determined to be 5 µM and
15.9 µmol/min/mg, respectively. These enzymatic para-
meters differed between enzymes from different species
(Table 1). The Km and Vmax values of tHBPA hydratase-
aldolase from P. fluorescens N3 for 2-hydroxybenzalde-
hyde were recently reported as 3.58 mM and 35.46
μmol/min/mg, respectively [2]; however, these values
were for the 2-hydroxybenzaldehyde and not tHBPA.
In addition, Ohmoto et al. determined the Km values of
two purified tHBPA hydratase-aldolases (HA A and HA
B) from S. pucimobilis TA-2, which were found to be 9
(HA A) and 3 (HA B) μM, respectively [6].
Subsequently, the specificity constants kcat and kcat/Km
of RnoE were determined using tHBPA as a substrate.
We determined the kcat and kcat/Km values of RnoE as
being 0.24 s−1 and 12.2 s−1 mM−1, respectively. These
values differed from those of P. aeruginosa Pak1 (kcat,
0.03 s−1 and kcat/Km, 6.0 s−1 mM−1). Although the Km,
kcat and kcat/Km values of RnoE is higher than that of
PahE, the specificity constants of other hydratase-
aldolases have not been reported.
Although RnoE catalyzes the aldol reaction of
tHBPA to 2-hydroxybenzaldehyde and pyruvate
(Figure 2(a)), its activity was investigated using 2-
chlorobanzaldehyde, 3-chlorobanzaldehyde, 4-chlor-
obanzaldehyde, and 2-fluoro-3-(Trifluoromethyl)ben-
zladehyde as substrates using HPLC. The substrate
peak areas decreased, and condensation products
were produced with all these halogenated substrates
(Figure 2(b-e)), indicating that RnoE can catalyze the
formation of various α, β-unsaturated ketones from
aldehyde and pyruvate. The conversion from fluori-
nated benzaldehyde and pyruvate to α, β-unsaturated
ketones by RnoE in this study is a remarkable cata-
lytic reaction by hydratase-aldolase because there
have been no reports of fluorinated benzaldehyde
being a substrate of aldol reaction by hydratase-
aldolase.
In conclusion, RnoE can catalyze both aldol and
retro-aldol reactions using benzaldehydes and pyr-
uvate as substrates. We believe that this is the first
report to show that fluorinated benzaldehyde can be
a substrate for aldol reaction by hydratase-aldolase.
Aldol condensation is generally performed under
acidic or alkaline conditions; however, RnoE could
catalyze the reactions at neutral pH and relatively
high temperatures. Therefore, various chemical con-
versions might be possible using this enzyme,
although further characterization is needed to
achieve this goal.

4
T. SUZUKI AND N. TAKIZAWA
a)
b)
c)
2
4
6
1
5
3
6
4
2
200
450
200
450
200
450
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
0
7.5
15
0
5
10
15
20
0
5
10
15
20
Retention time (min)
Retention time (min)
Retention time (min)
d)
e)
10
8
10
7
9
200
450
Wavelength (nm)
200
450
8
Wavelength (nm)
0
5
10
15
20
0
5
10
15
20
Retention time (min)
Retention time (min)
Figure 2. HPLC chromatograms from in vitro aldol reactions with various-halogenated banzaldehydes and pyruvate catalyzed by
RnoE.
(a) Production of trans-o-hydroxybenzylidenepyruvate (peak 1) from 2-hydroxybenzaldehyde (peak 2) and pyruvate. Retention time in this reaction is
short because this reaction was separately performed from reactions b–e. (b) Production of o-chlorobenzilidenepyruvate (peak 3) from 2-chloroban-
zaldehyde (peak 4) and pyruvate. (c) Production of m-chlorobenzilidenepyruvate (peak 5) from 3-chlorobanzaldehyde (peak 6) and pyruvate. (d)
Production of p-chlorobenzilidenepyruvate (peak 7) from 4-chlorobanzaldehyde (peak 8) and pyruvate. (e) Production of 2-fluoro-3-(Trifluoromethyl)
benzilidenepyruvate (peak 9) from 2-fluoro-3-(Trifluoromethyl) benzladehyde (peak 10) and pyruvate. In all chromatograms, upper panels represent
reactions without RnoE and lower panels represent reactions with RnoE. Each reaction mixture (Total volume 3.0 ml) contains 100 mM sodium pyruvate,
10 mM benzaldehydes, and 0.2 mg RnoE. All enzyme reactions were performed at 60°C for 60 min.
Acknowledgments
References
We would like to thank Professor emeritus Hohzoh Kiyohara
(Okayama University of Science) for their advice. The authors
would like to thank Enago (www.enago.jp) for the English
language review.
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Disclosure statement
No potential conflict of interest was reported by the authors.

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