Investigation of a New Type I Baeyer–Villiger Monooxygenase from Amycolatopsis thermoflava Revealed High Thermodynamic but Limited Kinetic Stability

Article abstract of DOI:10.1002/cbic.201900501

Baeyer–Villiger monooxygenases (BVMOs) are remarkable biocatalysts, but, due to their low stability, their application in industry is hampered. Thus, there is a high demand to expand on the diversity and increase the stability of this class of enzyme. Sta

Full text of DOI:10.1002/cbic.201900501

Accepted Article
Title: Investigation of a Novel Type I Baeyer-Villiger Monooxygenase
from Amycolatopsis thermoflava Revealed High Thermodynamic-
but Limited Kinetic Stability
Authors: Hamid R. Mansouri, Marko D. Mihovilovic, and Florian Rudroff
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10.1002/cbic.201900501
ChemBioChem
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Investigation of a Novel Type I Baeyer-Villiger Monooxygenase from Amycolatopsis
thermoflava Revealed High Thermodynamic- but Limited Kinetic Stability
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Hamid R. Mansouri1, Marko D. Mihovilovic1, Florian Rudroff1*
1Institute of Applied Synthetic Chemistry, TU Wien, Getreidemarkt 9, 1060, Vienna, Austria
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Abstract
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Baeyer-Villiger monooxygenases (BVMOs) are remarkable biocatalysts but due to their low
stability, their application in industry is hampered. Thus, there is a high demand to expand the
diversity and increase the stability of this class of enzyme. Starting from a known thermostable
BVMO sequence from Thermocrispum municipale (TmCHMO), we identified a novel BVMO
from Amycolaptosis thermoflava (BVMOFlava) that was successfully expressed in Escherichia coli
BL21(DE3). We investigated the activity and stability of the purified enzyme and assigned the
substrate profile for structurally different cyclohexanones and cyclobutanones. The enzyme
showed lower activity in comparison to cyclohexanone monooxygenase (CHMOAcineto) from
Acinetobacter sp. as the prototype BVMO but indicated higher kinetic stability by showing 2-fold
increased half-life at 30 °C. The thermodynamic stability, represented by the melting temperature,
resulted in a Tm value of 53.1 ℃ for BVMOFlava that is comparable to the Tm of TmCHMO
(Tm = 1°C) and significantly higher than the Tm value for CHMOAcineto ((Tm = 14.6°C)). We
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observed a strong deviation between the thermodynamic and the kinetic stability of BVMOFlava
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which might has a major impact for future enzyme discovery of BVMOs and their synthetic
applications.
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Keywords: BVMO, In silico, Substrate profile, Enzyme stability, Enzyme discovery
Introduction
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Baeyer-Villiger Monooxygenases (BVMOs) have been identified, isolated and characterized in the
late 1960s and since then became highly versatile biocatalysts for the oxidation of ketones and
aldehydes into the corresponding esters or lactones (Baeyer-Villiger reaction).[1] These enzymes
utilize molecular oxygen, operate at ambient temperatures and under slightly basic conditions,
while the conventional chemical reactions often require explosive and hazardous oxidants like
peracids.[2] Based on the type of cofactor accepted by the enzyme, two different BVMO types can
be classified. Type I is tightly bound to flavin adenine dinucleotide (FAD) as a cofactor and uses
NADPH as a source of electrons, while type II relies on flavin mononucleotide (FMN) and uses
NADH as an electron donor.[3] Type I BVMOs catalyze the oxidation of ketones into esters or
lactones [4, 5] with exceptional high regio-, chemo- and enantioselectivity for the production of fine
chemicals or chiral building blocks.[2a, 6] Based on these features, many industrial applications[7]
have been suggested but due to low operational stability[8] under given reaction conditions,
exploitation on a large scale is still challenging.[9] Researchers attempted to overcome this
limitation applying different approaches, such as reaction engineering[10], protein engineering[8, 11]
and metagenome mining[12]. For example, Goncalves and co-workers could increase the kinetic
stability of CHMOAcineto 1000-fold by performing reaction engineering. They used a combination
of redox cofactors (NADPH and FAD) and natural catalytic antioxidants like superoxide dismutase
and catalase to stabilize the enzyme. The only industrial application for BVMOs was published by
Bong et al.[13], which is catalyzing the final step of esomeprazole synthesis by a heavily mutated
BVMO variant (41 mutations). A different strategy is based on in silico methods by sequence
similarity analysis. By exploring metagenomes, it is possible to find new BVMOs, which may
show higher stability and a broad substrate acceptance while avoiding tedious protein engineering.
One of the most stable BVMOs to date, phenylacetone monooxygenase (PAMO) from
thermophilic actinomycete Thermobifida fusca was found by this method.[12b] Recently, genome
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mining also guided Mattevi and co-workers to find two other thermostable cyclohexanone
monooxygenases (TmCHMO & PockeMO), which were isolated from thermophilic bacteria.[12a,
14]
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Intrigued by the work of Romero et al, we aimed to find a novel BVMO with altered
thermodynamic stability but maintained high activity and substrate acceptance based on sequence
similarity in silico approach. We envisaged exploiting the sequence space of thermophilic bacteria
by using the TmCHMO sequence as a starting point. Among sequences found in the NCBI
databank, a new putative BVMO sequence from the thermophilic organism Amycolatopsis
thermoflava, which was isolated from a heat-treated soil,[15] was selected. The enzyme was cloned
and expressed successfully, enzyme activity and stability (kinetic and thermodynamic) were
measured, and the substrate profile of this novel BVMO was investigated.
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Result and Discussion
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First, we blasted against the NCBI database using TmCHMO as search query, which is one of the
most thermostable BVMOs to date. The most similar sequence to TmCHMO among thermophilic
bacteria was selected and identified as a new putative BVMO from Amycolaptosis thermoflava
(BVMOFlava). The sequence similarity between BVMOFlava and TmCHMO was 83 % and contained
the conserved consensus (G/AGxWxxxxF/YPG/MxxxD and FxGxxxHxxxWP/D) of the Type I
BVMO family. Moreover, both Rossmann-fold motifs (GxGxxG/A), which are responsible for
dinucleotide binding were identified in BVMOFlava. The full alignment is depicted in the supporting
information (Fig S1).
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Furthermore, we performed a phylogenetic tree analysis with BVMOFlava. The phylogenetic tree
was constructed by PhyML (Fig 1) and visualized by TreeDYN to find the position of BVMOFlava
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between different groups of BVMOs (Fig 1). The mid-point rooted maximum likelihood
phylogram shows the diversity of different BVMOs from different groups, I to VII.[21] As can be
seen in the maximum likelihood phylogram (Fig 1), the sequence of BVMOFlava is close to the
sequence TmCHMO with a strong bootstrap statistical support of 100 %. The tree also shows that
BVMOFlava is placed in the clade of the CHMO-family and especially it is a close neighbour of
CHMOAcineto. This suggests BVMOFlava to display a similar substrate profile compared to
CHMOAcineto. A closer look at the structure of BVMOFlava (based on the sequence homology
towards TmCHMO) revealed a high similarity to TmCHMO, whereas for CHMOAcineto small
differences especially in the outer regions and some loops were observed (see SI Fig S2). This
could be an indication that the flexibility of BVMOFlava is hampered and therefore structural
stability could be increased.
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Fig 1 The sequences of recombinantly expressed BVMOs have been used to make the
phylogenetic tree, which was constructed by PhyML and visualized by Inkscape. Different
BVMOs are color-coded based on their group they belong. Group 1 (light blue), group 2 (pink),
group 3 (maroon), group 4 (blue), group 5 (green), group 6 (orange), and group 7 (violet).
BVMOFlava is located in group 3 (red). The accession code of the sequences can be found in the
supporting information (Table S1).
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Expression and Purification
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With this novel putative type I BVMO sequence in hand we ordered the synthetic gene already
cloned into a pET22b(+) expression vector with a His-Tag on the C-terminus. Subsequently E. coli
BL21 DE(3) was transformed and protein expression was performed in the presence of isopropyl-
β-D-thiogalactopyranoside (IPTG, 50 μM) at 20 °C for 20-22 h. Successful expression was
analysed by SDS-PAGE analysis (Fig S3). A 59.5 kDa band of the purified enzyme was found that
belong to the new BVMOFlava. Purification was performed by standard His-Trap affinity column.
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Activity and Stability measurement:
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After successful soluble protein expression and purification, we investigated the activity and stability of
BVMOFlava. First, we had to find a suitable substrate, since the natural one was unknown. Based on the
sequence similarity to CHMOAcineto we assumed a comparable substrate profile and tested
cyclohexanone as a model compound. Indeed, cyclohexanone was converted to the corresponding
- caprolactone. The activity was comparable to TmCHMO and approximately 10-fold lower than
CHMOAcineto (Fig 2). Kinetic values have been studied for all three enzymes. The Km value for
cyclohexanone and BVMOFlava was 0.53±0.1 µM, for TmCHMO it was below <1 μM[22] and for
CHMOAcineto it turned out to be one order of magnitude higher and gave 6.74±2 μM. Next, we
determined kcat, which resulted in the highest value for CHMOAcineto 15±1.3 s-1 in comparison to
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BVMOFlava1.5±0.1 s- 1 and TmCHMO 2.0 s-1 (see supporting information Table S2). In thefollowing,
we investigated the pH optimum, the temperature profile, thermodynamic- and kinetic stability of
BVMOFlava in comparison to CHMOAcineto and TmCHMO. The optimum pH for the activity was
measured at different pH values ranging from 7.5 to 10.5 with an interval of 1 (Fig. 2a). BVMOFlava
showed the highest activity at pH 7.5, CHMOAcineto has its optimum at 8.5 whereas TmCHMO is
equally active from 7.5-9.5 (Fig. 2a). Especially at higher pH values (10.5), TmCHMO outperforms
BVMOFlava and CHMOAcineto maintaining 50% of its initial activity. Next, we determined the
temperature optimum for all three enzymes, which turned out to be 45 ℃ for both BVMOFlava and
CHMOAcineto while TmCHMO showed the highest activity at 60 ℃ (Fig 2b). This result is in
contrast to our expectations, since the sequence of BVMOFlava originated from a thermophilic
organism. A different picture was observed by comparison of the thermodynamic stability by their
melting temperatures (Tm, Fig 2c). BVMOFlava showed the highest Tm (53.1±0.2 ℃) whereas
TmCHMO and CHMOAcineto showed a Tm of 52.1±0.6 °C and 38.5±0.1 ℃, respectively (Fig 2c).
This finding is in agreement with the origin of the sequence based on the thermostable TmCHMO.
Interestingly, TmCHMO showed a second transition midpoint that might indicates an unfolding
and deactivation process with two active native states. When the temperature exceeds the second
limit, the enzyme goes into the unfolded and deactivated state.
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Next, we investigated the kinetic stability (half-life = t1/2) of all three BVMOs at 30 ℃, 40 °C and
60 °C (for detailed reaction conditions see material and methods). At 30 °C, BVMOFlava is
approximately 2-fold (73±10 min) more stable than CHMOAcineto (46±6 min) and 7.5-fold less
stable than TmCHMO (549±51 min, Fig 2d). A similar picture was observed after incubation for
1 h at 40°C. CHMOAcineto showed a t1/2 for 2.02±0.45 min, whereas BVMOFlava is 3-times more
stable with a t1/2 of 6.00±0.80 min. In contrast, TmCHMO still shows more than 60 % of its residual
activity. We investigated the t1/2 of TmCHMO at 60 °C and determined a half-life of 0.8±0.2 min
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(supporting information figure S5A and S5B). These results confirmed also our previous finding
that thermodynamic stability did not necessarily correlate to kinetic stability within BVMO
biocatalysts.[10a] Moreover, we also investigated the stability of all three enzymes in presence of
different organic solvents (5 % v/v, Fig 2e). All of them showed a decent stability in the presence
of MeOH, whereas 5 % v/v ACN affects CHMOAcineto the most and resulted in almost complete
loss of activity. The most destructive co-solvent for the investigated enzymes was THF, which led
immediately to the complete deactivation of all three enzymes.
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Fig 2a) Effect of pH on activity at 30 °C, 50 mM TrisHCl + 10 µM FAD, 0.5 mM cyclohexanone and
100 µM NADPH. b) Activity measurement at various temperatures from 30 to 70 ℃ (same conditions as
for the pH study). c) Melting temperature determination was performed by nano differential scanning
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fluorimetry (nanoDSF): 50 mM TrisHCl, 10 µM FAD, 2 mg mL-1 enzyme. d) Half-life measurement:
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incubation at 30 °C, 10 µM enzyme, 50 mM TrisHCl, 10 µM FAD, pH 7.5. e) Determination of half-life in
the presence of 5 % co-solvent (same condition as for half-life measurement).
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Substrate Profile of BVMOFlava
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After full biochemical characterization of the novel BVMOFlava we elucidated its substrate profile for
potential industrial applications and compared it to literature data from CHMOAcineto and TmCHMO.
We applied whole-cell biotransformations under non-growing conditions and analysed the performance
(conversion and enantiomeric excess) by chiral gas chromatography after 24 h reaction time. Positive
control experiments were performed with cyclohexanone. First, Baeyer-Villiger oxidation of different
substituted cyclohexanones and cyclobutanones were studied.
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The substrate acceptance and enantiopreference of 4-substituted cyclohexanones 1a-d are quite
comparable for all three enzymes (Table 1). The only minor exception was found for the bulky
substrate 1c which gave full conversion in the presence of BVMOFlava and a very high optical purity
(96 % ee) of the desired lactone (Table 1). A similar trend was observed for substrates 2 and 3,
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whereas all three enzymes showed the same catalytic performance with respect to conversion and
enantiopreference.
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The kinetic resolution of 4a and 4b resulted in formation of the R enantiomer in up to 99 % ee after
almost 50 % conversion. Next, four different cyclobutanones were tested (5-7), whereas again same
conversions and enantioselectivities were obtained. Compounds 5a and 5b were poorly accepted
by CHMOFlava: substrate 5a gave almost racemic lactone whereas 5b resulted in the desired lactone
with 77 % ee optical purity. For the fused cyclobutanone 6 full conversion and perfect optical
purities for both the normal and the abnormal lactone were achieved. The normal lactone is an
intermediate in the synthesis of Corey lactone which is a building block for prostaglandin
synthesis[23] and the abnormal product is a starting material of brown algae pheromone synthesis[2a]
.
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Table 1 Baeyer-Villiger reaction of substituted cyclic ketones.
Substrate
R
BVMOFlava
ee (%)B
Reference reaction CHMOAcineto
Reference reaction TmCHMO
Conv (%)A
Conv (%)
ee (%)
98 (S)[24]
10 (R)[25]
>98 (S)[25]
60 (-)[26]
Conv (%)
ee (%)[22]
99 (S)
18 (R)
93 (S)
88 (-)
1a R=Me
1b R=OH
1c R=tBu
1d R=Ph
>99
83
99 (S)
8 (R)
>99
81
>99
89
>99
34
96 (S)
89 (-)
17
>99
30
82
2
3
59
99 (4S,6R)
85
99 (4S,6R)[27]
84
99 (4S,6R)
>99
P:D/41:59C
>99(-),96(-)
>99
P:D/49:51
>99
P:D/49:51
99(-),99(-)
99(-),99(-)[28]
4a R=Ph
4b R=Bn
40
42
94 (R)
99 (R)
41
38
98 (R)[28]
96 (R)[28]
49
48
97 (R)
98 (R)
5a R=Ph
5b R=Cl-
Ph
10
16
17 (R)
77 (S)
94
83
62 (R)[26]
81 (S)[26]
>99
81
49 (R)
95 (S)
6
>99
>99
N:ABN/50:50D
>99(-),>99(-)
>99
>99
N:ABN/51:49
95(-),>99(-)[28]
>99
>99
N:ABN/50:50
>99(-),>99(-)
7
N:ABN/59:41
72(-), >99(-)
N:ABN/65:35
60(-),>95(-)[28]
N:ABN/55:45
79(-),>98(-)
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ARelative conversion (Conv) of substrate to product
BEnantiomeric excess (ee) of product
CP:D ratio of proximal to distal lactone
DN:ABN ratio of normal to abnormal lactone
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Within our study, we identified a novel type I BVMO based on a sequence similarity search of the
recently published and thermostable TmCHMO. The BVMO from Amycolaptosis thermoflava
showed a high amino acid sequence similarity to TmCHMO with a molecular weight of 59.5 kDa.
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Based on the phylogenetic tree analysis, BVMOFlava belongs to group III of type I BVMOs and is
located in the same clade as CHMOAcineto and TmCHMO. As expected a similar substrate profile
for all three enzymes was determined. Although BVMOFlava originates from a thermophilic
organism the kinetic stability at slightly elevated temperatures dropped from 72 min (30 °C) to
6 min at 40 °C. In contrast, the thermodynamic stability (Tm-value) was quite comparable to
TmCHMO and significantly higher than the Tm of CHMOAcineto. The deviation between kinetic and
thermodynamic stability is a major problem in the field, since often only Tm values are published
without any context to the actual operational performance of the new catalyst. Within this study,
we would like to emphasize how important it is to determine both stabilities for future comparison
and putative industrial applications of BVMOs.
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Material and Methods
Materials
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All chemicals and reagents were from commercial sources (New England BioLabs, Ipswich,
Massachusetts, United States; Promega Corp., Madison, WI, USA; Carl Roth, Karlsruhe,
Germany; Lab M limited, Lancashire, United Kingdom; Sigma-Aldrich Corp., St. Louis, MO,
USA; Merck KGaA, Darmstadt, Germany; Chem Lab, Zedelgem, Belgium). All substrates in this
study either supplied commercially or synthesized in our lab. Distilled solvents have been used in
this study.
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Sequence analysis
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Multiple sequence alignment has been performed by MUSCLE (Multiple Sequence Comparison
by Log-Expectation).[29] The phylogenetic tree was generated by using phylogeny.fr.[30] The
homology model was created by SWISS-MODEL[31] and protein 3D structure visualized by Swiss
PDB viewer.[32] Multiple structure alignment has been carried out by PyMOL.
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Plasmid construction, microbial strains and culture media
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An optimized DNA fragment containing the selected BVMO genes from
A. thermoflava (WP_027929099.1) and TmCHMO (WP_028849141.1) were synthesized and
inserted into pET22b(+) by GeneScript with NdeI and NotI restriction sites, respectively.
CHMOAcineto was obtained from G. Chen et al[33] The synthesized gene was confirmed by
sequencing using T7 and T7term primers. E. coli strain BL21 (DE3) was transformed by heat-
shock using standard procedures provided by neb transformation kit. Transformed cells were
grown in an incubator operating at 37 °C in lysogeny broth–agar (LB-agar) medium supplemented
with 100 μg mL−1 ampicillin.
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Protein expression
E. coli strain BL21(DE3) was used as an expression host for all enzymes in this study (BVMOFlava
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,
CHMOAcineto and TmCHMO). LB medium (5 mL) supplemented with ampicillin to a final
100 μg mL−1
BL21(DE3)
concentration
of
was
inoculated
with
E. coli
pET22b(+)_BVMOFlava/CHMOAcineto/TmCHMO and incubated in an orbital shaker at 37 °C,
200 rpm over-night. Pre-cultivated bacteria (2 % v/v) was transferred to a 1 L flask containing
250 ml LB with the same concentration of ampicillin as before. They were incubated at 37 °C,
200 rpm for 2 hours to reach an optical density between 0.6-0.8 at 590 nm. Then Isopropyl
β- d- thiogalactopyranoside (IPTG) was added to the final concentration of 50 μM and the flask
was transferred to 20 °C and incubated for 18-22 h.
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Enzyme purification
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All further steps were carried out at 4 ºC to protect the enzyme against inactivation. The overnight
culture containing expressed recombinant cells were centrifuged at 8000 x g, 4 ℃ for 10 min and
cells were collected. Cell pellets were resuspended in 50 mM TrisHCl, pH 7.5 containing 100 μM
FAD and 100 μM of PMSF (phenylmethylsulfonyl fluoride). The crude cell extract was sonicated
by a Bandelin KE76 sonotrode connected to a Bandelin Sonoplus HD 3200 in 9 cycles (5s pulse,
55s break, amplitude 50 %). Cell debris and aggregates were removed by centrifugation
(25000 x g, 25 min, 4 ºC, JA-17 Beckmann rotor). Supernatant was filtered using a 0.25 μm filter,
equilibrated with 50 mM TrisHCl, pH 7.5, 0.5 M NaCl, 100 μM FAD and applied on 1 mL of
Ni- sepharose column (1 mL, GE Healthcare Bioscience). The unwanted non-attached proteins
were washed by using 5 column-volumes of 50 mM TrisHCl, 0.5 M NaCl, 40 mM imidazole,
100 μM FAD, at pH 7.5. Elution step has been performed by applying 5 column-volumes of 50 mM
TrisHCl, 0.5 M NaCl at pH 7.5 containing 400 mM imidazole and 100 μM FAD. The eluted
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enzymes were washed by 50 mM TrisHCl, 100 μM FAD, at pH 8 and concentrated with an
ultracentrifugal tube with a cut-off of 10 kDa.[10a]
Activity and Stability measurement
Enzyme activity was measured by monitoring the decrease of NADPH absorbance at 340 nm.
Standard assays contained the enzyme (0.05 μM), cyclohexanone (0.5 mM) and NADPH (100 μM)
in 50 mM TrisHCl, adjusted to the desired pH. All the measurement have been carried out at 30 °C
[10a]. The reaction was started immediately after enzyme addition by mixing 4 μL NADPH (25 mM
stock solution) to the cuvette (final volume 1 mL). Oxidation of NADPH was followed at 30 °C in
a Lambda 35 spectrophotometer (Perkin–Elmer, Waltham, MA, USA) for 120 seconds. Stability
measurement was performed by incubating 10 μM enzyme at 30 °C in 50 mM TrisHCl, 10 μM
FAD, pH 7.5. Samples were taken at different time points and added to a cuvette containing
100 μM NADPH and 0.5 mM substrate to test for catalytic activity. The stability in presence of
different co-solvents (MeOH, ACN and THF) with final concentration of 5 % was measured under
the same reaction conditions as described before. The experimental data were fitted to an
exponential decay equation using Origin Pro software (Origin 9.1 for Windows). The regression
data are depicted in the SI.
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Melting temperature measurement
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The melting temperatures (Tm) of all three enzymes were measured by Prometheus NT.48. The
samples were prepared in TrisHCl 50 mM pH 7.5, 10 µM FAD with a final enzyme concentration
of 2 mg mL-1 and the samples run from 20 to 95 °C.
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Biotransformations
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Recombinant protein expression was performed in LB medium, supplemented with ampicillin
(100 μg mL−1). The enzyme expression was induced by IPTG (final concentration of 50 µM) at
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20 °C. Cells were centrifuged (8000 x g, 4°C, 10 min) and resuspended and washed in 50 mM PBS
buffer pH 7.4. After washing, the cells were centrifuged (8000 x g, 4 °C, 10 min) and resuspended
again with the same buffer to reach OD 30. 1 mL (OD590=30) of recombinant expressed cells
suspended in PBS buffer (pH 7.4, 50 mM), and 10 mM substrate final concentration, (methanol as
co-solvent (5 % of total volume)) The components of the reaction (1.02 mL in total) were added
into a 25 mL flask, and the reaction was performed at 30 °C by shaking (220 rpm) for 24 h.[12a] The
product was extracted with ethyl acetate containing 0.1 mM methyl benzoate as the internal
standard for the GC analysis. The product analysis was performed with GC (Thermo Scientific
Trace or Focus GC, Thermo Fisher Scientific, Waltham, MA, USA) using chiral/achiral column.
Product validation was performed by literature known reference biotransformations. The
information of columns and methods for the GC experiments are listed in the supporting
information.
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Acknowledgement
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Financial support by TU Wien ABC-Top Anschubfinanzierung, Austrian agency for international
cooperation in education and research (OeaD) and WWTF Project LS17-069 are gratefully
acknowledged.
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