Self-sufficient Baeyer-Villiger monooxygenases: Effective coenzyme regeneration for biooxygenation by fusion engineering

Article abstract of DOI:10.1002/anie.200704630

(Chemical Presented) Two-in-one biocatalysts were engineered by the covalent fusion of NADPH-dependent Baeyer-Villiger monooxygenases to a phosphite dehydrogenase for coenzyme regeneration (see scheme). Not only the purified fusion proteins, but also whole cells and crude cell extracts containing the enzyme conjugates, could be used to catalyze biotransformations with high efficiency. NADP⁺=nicotinamide adenine dinucleotide phosphate.

Full text of DOI:10.1002/anie.200704630

Angewandte
Chemie
DOI: 10.1002/anie.200704630
Biocatalysis
Self-Sufficient Baeyer–Villiger Monooxygenases: Effective Coenzyme
Regeneration for Biooxygenation by Fusion Engineering**
Daniel E. Torres Pazmiæo, Radka Snajdrova, Bert-Jan Baas, Michael Ghobrial,
Marko D. Mihovilovic,* and Marco W. Fraaije*
Over the past few years, industrial interest in biocatalysts that
perform selective oxidative reactions has increased signifi-
cantly.[1] Baeyer–Villiger monooxygenases (BVMOs) have
been identified as a highly versatile class of enzymes for the
efficient catalysis of chemo-, regio-, and/or enantioselective
oxygenation reactions.[2] Although the most prominent trans-
formation catalyzed by these biocatalysts is a chiral variant of
the classical Baeyer–Villiger reaction,[3,4] the oxygenation of
heteroatoms and epoxidation reactions have also been
reported.[5] Stoichiometric amounts of O2 and NADPH are
required for these reactions. A complication for the large-
scale application of these reactions is the high cost of the
reduced nicotinamide coenzyme.[6] To overcome this prob-
lem, several electrochemical and photochemical approaches
have been explored.[7] However, the efficiency of these
approaches is typically poor. Furthermore, it has been
shown that BVMOs require NADP+ for stability and
enantioselective catalysis.[8]
An efficient and commonly used method for coenzyme
regeneration employs whole cells, especially in combination
with the recombinant expression of the required biocata-
lysts.[9] This strategy has been implemented in BVMO-
mediated biotransformations with wild-type strains[10] and
has proved particularly successful with recombinant over-
expression systems.[11,12] The approach avoids laborious
enzyme purification steps and exploits the coenzyme regen-
eration capacity of the host. Although whole cells have been
shown to be effective catalysts for Baeyer–Villiger oxida-
tion,[13] they also exhibit limitations, such as cellular toxicity,
enzyme inhibition by the substrate/product, degradation of
the product, and poor oxygen-transfer rates.[14] Coenzyme
regeneration by using isolated enzymes has also been studied
extensively in the past few years.[15] Well-known examples of
such NADPH-regenerating enzymes are alcohol dehydrogen-
ase and formate dehydrogenase.[16] A phosphite dehydrogen-
ase (PTDH) was also identified as an effective enzyme for
coenzyme regeneration.[17] The favorable thermodynamic
equilibrium constant makes the oxidation of phosphite a
nearly irreversible process.[18] The exquisite selectivity of
PTDH for phosphite also precludes any side reactions, such as
those that can occur, for example, when an alcohol dehydro-
genase is used. These characteristics make PTDH an ideal
candidate for use as a coenzyme regenerating enzyme (CRE)
in combination with BVMOs or other NAD(P)H-dependent
enzymes.
Herein, we report a novel approach to the combination of
the catalytic activity of a redox biocatalyst with concomitant
coenzyme recycling in a single fusion protein (Scheme 1).
During the last decade, a number of fusion protein tags have
been developed. These tags are used intensely in life-science-
related research and commercial activities. Although the
fusion of proteins is a widely applied strategy in, for example,
enzyme purification (e.g. the use of glutathione S transferase
(GST) tags)[19] and the subcellular visualization of target
proteins (e.g. with a green fluorescent protein (GFP) tag),[20]
this concept is hardly ever encountered in the context of
synthetic applications. Only a few isolated examples in the
literature provide evidence that the fusion of separate
enzymes can result in improved biocatalytic properties.[21]
We report herein on the engineering of a number of
representative BVMOs that are linked covalently to soluble
NADPH-regenerating phosphite dehydrogenase. This con-
struct enables the use of phosphite as a cheap and sacrificial
electron donor with whole cells, cell extracts, and purified
enzyme. It was our particular goal to design a self-sufficient
two-in-one redox biocatalyst that does not require an addi-
tional catalytic entity for coenzyme recycling. As model
[*] Dr. R. Snajdrova, M. Ghobrial, Prof. Dr. M. D. Mihovilovic
Institute ofApplied Synthetic Chemistry
Vienna University ofTechnology
Getreidemarkt 9/163-OC, 1060 Wien (Austria)
Fax: (+43)1-58801-15420
E-mail: mmihovil@pop.tuwien.ac.at
D. E. Torres Pazmiæo, J. B. Baas, Prof. Dr. M. W. Fraaije
Laboratory ofBiochemistry
Groningen Biomolecular Sciences and Biotechnology Institute
University ofGroningen
Nijenborgh 4, 9747 AG Groningen (The Netherlands)
Fax: (+31)50-363-4165
E-mail: m.w.fraaije@rug.nl
[**] We thank CERC3 and the Austrian Science Fund (FWF project no.
I19-B10) for funding, and W. A. van der Donk, University of Illinois,
for providing the pRW2 double-mutant template. This study was
conducted in association with COST action D25 - Applied
Biocatalysis within the working group “Biooxidation”.
Supporting information for this article, including experimental
details, is available on the WWW under http://www.angewandte.org
or from the authors.
Scheme 1. Coenzyme regeneration by CRE/BVMO fusion enzymes.
NADPH is the reduced form of nicotinamide adenine dinucleotide
phosphate (NADP+).
Angew. Chem. Int. Ed. 2008, 47, 2275 –2278
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Table 2: Conversion ofphenylacetone (PA) by either PAMO–CRE
(0.2 mm) or PAMO (0.13 mm) in the presence ofPTDH upon incuba-
tion at 308C for 3 h.
BVMOs, we selected thermostable phenylacetone mono-
oxygenase (PAMO) from Thermobifida fusca,[22] well-studied
cyclohexanone monooxygenase (CHMO) from Acinetobacter
sp.,[23] and cyclopentanone monooxygenase (CPMO) from
Comamonas sp.[24] To overexpress such CRE/BVMO fusion
proteins, we constructed expression vectors (pCRE) that
result in the expression of BVMOs fused to PTDH by a short
linker peptide. We formed two fusion enzymes with PAMO by
linking PAMO to the N or C terminus of PTDH, whereas
CHMO and CPMO were linked to PTDH at their N terminus.
All four bifunctional enzymes showed excellent expression
levels when E. coli TOP10 was used as the host. The CRE/
BVMO enzymes were purified by column chromatography to
yield 10–50 mg of pure and soluble CRE/BVMO from 1 L of
culture broth, with the exception of CRE–CPMO, which has
never been purified successfully from a recombinant host.[24]
Steady-state kinetic analysis of the CRE/BVMO enzymes
revealed that the fusion of the BVMOs with PTDH hardly
affected their respective catalytic properties at all (Table 1).
The rates of catalytic activity (kcat) of the BVMO subunits
were similar to those observed for the separate enzymes. The
only significant effect observed was a decrease in the affinity
of PTDH for phosphite (the KM value is approximately 13
times higher) when fused to a BVMO. Nonetheless, the
observed kinetic parameters should allow efficient conver-
sions.
[PA]
[mm] [mm]
[NADP+] TOF[a]
[hÀ1
TTN[b]
TTN[b]
]
(PAMO) (NADP+)
PAMO–CRE
2.5
5
5
125
3.3”103 9.9”103 395
4.5”103 1.4”104 360
PAMO and PTDH 2.5
PAMO and ADH[c] 7.5
98
4.7”103
12
[a] Turnover frequency, as described in Ref. [25]. [b] Total turnover
number with respect to the biocatalyst or the coenzyme, as described
in Ref. [25]. [c] Adapted from Ref. [15b].
probably as a result of the instability of the enzyme at low
concentrations of NADP+.
We also studied the applicability of these two-in-one
biocatalysts in whole cells and in crude cell extracts by
carrying out (enantioselective) transformations with CRE–
CHMO and CRE–CPMO. Initial screening experiments to
investigate any effect on stereoselectivity of the additional
PTDH domain within the bifunctional fusion protein were
carried out according to a previously established protocol
based on miniscale whole-cell biotransformations.[26] We
examined the desymmetrization of prochiral substrates,
which enables control over up to six stereogenic centers in a
single step (Scheme 2), as well as the regiodivergent bio-
oxidation of fused racemic cyclobutanones (Scheme 3), one of
the most remarkable transformations promoted by BVMOs
that oxidize cycloketones (see the Supporting Information).
We observed essentially no adverse influence from the
fusion of the two catalytic entities. In the majority of cases, the
efficiency of the biotransformation remained similar to that of
the equivalent transformation under the catalysis of the
original unfused enzymes (as indicated by comparable con-
versions within standardized reaction times; see the
Supporting Information). The stereoselectivity of the fused
biocatalysts was also largely comparable to that of the
individual enzymes, and in some cases (the formation of 21
with CRE–CHMO and 24 with CRE–CPMO) the product
lactones were formed with even better enantioselectivities in
the presence of the fusion enzymes. Only in very few cases
No inhibition of the activity of either the BVMO or
PTDH by the substrate or product of the other subunit was
observed. Activity measurements at elevated temperatures
showed that the PAMO and PTDH domains have similar
thermostabilities compared to the separate isolated
enzymes.[18,22] Moreover, we determined the NADPH-regen-
eration efficiency of PAMO–CRE by following the conver-
sion of phenylacetone (PA; 2.5 mm) with time. In the presence
of NADP+ at a concentration of only 5 mm, the bifunctional
fusion enzyme converted effectively phenylacetone into
benzyl acetate (79% conversion after 3 h). Control experi-
ments in which equal amounts of units of each the individual
enzymes PAMO and PTDH were added in place of the fusion
enzyme yielded similar results (Table 2). In similar experi-
ments carried out recently with PAMO in combination with
an alcohol dehydrogenase in a two-liquid-phase system, lower
TOF and total turnover number (TTN) values were found in
the presence of 125 mm NADP+.[15b] Strikingly, even in the
presence of equimolar amounts of NADP+ and PAMO–CRE
(0.2 mm), 13% conversion was observed, with a TTN of 1750
with respect to the regeneration of the coenzyme. Unfortu-
nately, we observed a decrease in catalytic activity with time,
Table 1: Kinetic parameters of the bifunctional fusion enzymes.[a]
PAMO–CRE
CRE–PAMO
CRE–CHMO
k
cat(ketone)
1.9 sÀ1 (70%)
52 mm (70%)
2.9 sÀ1 (100%) 14 sÀ1 (100%)
KM(ketone)
KM(NADPH)
k
53 mm (70%)
<1 mm (100%)
6.5 sÀ1 (340%)
3.5 mm (50%)
4 mm (60%)
<1 mm (100%)
cat(phosphite)
4.3 sÀ1 (225%)
2.6 sÀ1 (140%)
KM(phosphite) 285 mm (1400%) 317 mm (1500%) 228 mm (1100%)
KM(NADP+)
12 mm (340%) 15 mm (430%) 12 mm (340%)
[a] The relative kinetic parameters ofthe native enzymes are shown in
parentheses.[18,22,23]
Scheme 2. Types oflactone products obtained rfom stereoselective
desymmetrization reactions with CRE/BVMO fusion enzymes.
2276 www.angewandte.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2008, 47, 2275 –2278

Angewandte
Chemie
employing bifunctional fusion proteins: Our CRE/BVMO
concept in combination with the simple application protocol
as a cell extract offers a facile method to establish substrate-
acceptance profiles for enzymes with a minimum of effort in
terms of protein purification and maximum simplicity with
respect to coenzyme regeneration.
In conclusion, we have created a self-sufficient redox
biocatalyst by fusing two independent enzymes to form a new
bifunctional biocatalyst. The present study demonstrated the
feasibility of this novel concept for coenzyme regeneration
with three distinct Baeyer–Villiger monooxygenases and a
phosphite dehydrogenase for orthogonal coenzyme recycling.
The fused BVMOs are complementary in their substrate
profiles (PAMO accepts aromatic ketones, CHMO and
CPMO accept aliphatic cycloketones) and their stereoselec-
tivity (CHMO and CPMO produce the opposite lactone
enantiomers in a large variety of examples). The three
monooxygenases are sufficiently different in sequence and
in terms of their phylogenetic relationships to suggest the
general applicability of this coenzyme regeneration concept,
at least among the family of BVMOs. Considering the diverse
reactivity of novel members of this family,[31] our study may
contribute to the further proliferation of this highly interest-
ing biotransformation platform. Presently, we are conducting
additional studies to further optimize the efficiency of these
newly developed self-sufficient BVMOs for ultimate appli-
cation in the large-scale production of chiral intermediates for
Scheme 3. Regiodivergent CRE/BVMO-mediated oxidation ofuf sed
bicyclic ketones.
was erosion of optical purity observed in the formation of the
lactones (e.g. 12 with both CRE–CHMO and CRE–CPMO);
however, the absolute configuration of the products remained
unchanged. A more pronounced effect was found with the
series of prochiral cyclobutanones. We attribute this to the
largely different conformational energies on this ring system
compared to structurally better defined cyclopentanones and
-hexanones, as recently outlined.[27] Remarkably, we observed
two cases in which the substrate range of the original BVMO
was extended as a consequence of the fusion process (the
formation of 10 with CRE–CHMO and 28 with CRE–
CPMO). Although in both cases the catalytic efficiency was
limited, recently described strategies could be applied to
further improve performance.[28]
The behavior of the original biocatalysts was also very
similar to that of the fused CRE/BVMO enzymes for the
regiodivergent oxidation of fused cyclobutanones (see the
Supporting Information). Whereas CRE–CHMO yielded
equimolar amounts of the “normal” and “abnormal” lactones
with high ee values in most cases, CRE–CPMO produced
predominantly “normal” lactones, but with low stereoselectivity.
With respect to facile and rapid application of these new
and self-sufficient CRE/BVMOs in stereoselective synthesis,
we prepared a crude cell extract (CE) from CRE–CHMO
producing recombinant E. coli. The optimum concentrations
of both phosphite and 4-methylcyclohexanone as a model
substrate were determined (see the Supporting Information),
and the fusion biocatalyst as CE was demonstrated to
completely convert the ketone (5 mm) into the chiral lactone.
A particularly interesting aspect in the utilization of CRE/
BVMOs as CE is the fact that the preparative-scale biotrans-
formations outlined above were performed without addition
of NADP+, taking advantage of the coenzyme present in
E. coli cells ( ꢀ 200 mm).[29] Our observation that a relatively
low concentration of NADP+ is sufficient for catalysis
suggested that the amount of the nicotinamide coenzyme
liberated upon cell breakage should indeed enable effective
conversion with cell extracts containing overexpressed CRE/
BVMO (cell-pellet volume/incubation volume = 1:2; total
protein concentration ꢀ 20 mgmLÀ1). By using a cell extract
we also confirmed that the unsaturated bicycloketone pre-
cursor to 23 is a substrate for CRE–CHMO. (This biooxida-
tion was reported previously with isolated CHMO;[30a] how-
ever, when we tried to repeat the experiment by using a
recombinant whole-cell strain, only starting material was
recovered.[30b]) This result underscores a major advantage of
the synthesis of bioactive compounds.
Received: October 8, 2007
Published online: January 25, 2008
Keywords: asymmetric catalysis · Baeyer–Villiger reaction ·
.
biotransformations · coenzyme regeneration · fusion enzymes
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