Baeyer-Villiger monooxygenases: recent advances and future challenges.

Abstract

Baeyer-Villiger monooxygenases For many enzyme classes, a wealth of information on, for example, structure and mechanism has been generated in the last few decades. While the first Baeyer-Villiger monooxygenases (BVMOs) were already isolated more than 30 years ago, detailed data on these enzymes were lacking until recently. Over the last years several major scientific breakthroughs, including the elucidation of BVMO crystal structures and the identification of numerous novel BVMOs, have boosted the research on BVMOs. This has led to intensified biocatalytic explorations of novel BVMOs and structure-inspired enzyme redesign. This review provides an overview on the recently gained knowledge on BVMOs and sketches the outlook for future industrial applications of these unique oxidative biocatalysts.

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Available online at www.sciencedirect.com
Baeyer–Villiger monooxygenases: recent advances and future
challenges
Daniel E Torres Pazmin ˜o, Hanna M Dudek and Marco W Fraaije
Baeyer–Villiger monooxygenases
For many enzyme classes, a wealth of information on, for
example,structureandmechanismhasbeengeneratedinthe
last few decades. While the first Baeyer–Villiger
monooxygenases (BVMOs) were already isolated more than
30 years ago, detailed data on these enzymes were lacking
until recently. Over the last years several major scientific
breakthroughs, including the elucidation of BVMO crystal
structures and the identification of numerous novel BVMOs,
have boosted the research on BVMOs. This has led to
intensified biocatalytic explorations of novel BVMOs and
structure-inspired enzyme redesign. This review provides an
overview on the recently gained knowledge on BVMOs and
sketches the outlook for future industrial applications of these
unique oxidative biocatalysts.
Address
Laboratory of Biochemistry, Groningen Biomolecular Sciences and
Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG
Groningen, The Netherlands
Corresponding author: Fraaije, Marco W (m.w.fraaije@rug.nl)
Current Opinion in Chemical Biology 2010, 14:138–144
This review comes from a themed issue on
Biocatalysis and Biotransformation
Edited by Roger Sheldon and Jean-Louis Reymond
Available online 16th December 2009
1367-5931/$ – see front matter
# 2009 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.cbpa.2009.11.017
Baeyer–Villiger oxidation: chemical and
biochemical methods
In 1899, Adolf Baeyer and Victor Villiger described an
oxidation reaction in which carbonylic compounds are
converted into the corresponding esters or lactones.
Hence, this atypical reaction has been named Baeyer–
Villiger (B–V) oxidation. Nowadays, B–V oxidations are
frequently employed in synthetic organic chemistry [1,2].
However, because of the lack of selectivity of chemical
catalysts and the harsh conditions needed for catalysis,
the enzymatic counterparts are attractive to use as bio-
catalysts. The first Baeyer–Villiger monooxygenase
(BVMO) for which the synthetic potential was shown
is cyclohexanone monooxygenase (CHMO) [3]. While
CHMO is produced by the Acinetobacter bacterium for
catalyzing the first step in cyclohexanone degradation, it
has been shown to accept hundreds of different car-
bonylic compounds, displaying an exceptionally broad
substrate acceptance profile in combination with very
high regioselectivity and/or enantioselectivity [4,5?]. In
addition, further studies showed that various types of
oxidations could be catalyzed by CHMO and other
BVMOs [6–8]. These features render BVMOs as attrac-
tive oxidative biocatalysts. Efforts to identify and cata-
lytically exploit these enzymes have led in the last 10
years to the availability of new recombinant BVMOs for
biocatalysis (Figure 1). This review provides an overview
of the recent developments in discovery, structural, and
redesign studies on BVMOs. For more details on their
biocatalytic scope, we refer to recent reviews [9,10?].
Biodiversity of BVMOs
Along with the identification of several genes encoding
BVMOs at the beginning of this century [11–15], it was
realized that they form a sequence related enzyme family
(Type I BVMOs) of FAD-containing and NADPH-de-
pendent enzymes of which members can be identified
with a specific protein sequence motif [16]. Although the
molecular basis for the conservation of these specific
residues remained obscure until recently (vide infra),
the sequence motif has been very useful for mining
genomes for novel BVMOs. It has revealed a striking
distribution of these enzymes among organisms. While
bacteria are relatively rich in BVMOs, containing on
average one BVMO/genome, no Type I BVMOs are
present in human, plant, and animal genomes [9]. Among
bacteria, these enzymes are prevalent among actinomy-
cetes, rendering these bacteria an interesting source for
novel BVMOs while also fungal genomes are relatively
rich in BVMOs and unexplored. Recent studies on the
biotransformation of natural products confirm the vital
roles that BVMOs play in microbial metabolic pathways.
BVMOs catalyze key steps in the degradation of, for
example, acetone [17], bulky cyclic aliphatic ketones
[18], and linear ketones [19–21] (Figure 2A). Also highly
complex biomolecules are tailored by BVMOs as exem-
plified by the conversion of steroids [22,23] and biosyn-
thesis of sesquiterpenoids [24] and aflatoxins [25]
(Figure 2A). Except for being highly suitable for bioca-
talytic applications, BVMOs can also be of medical
relevance. The etaA gene of Mycobacterium tuberculosis
has been shown to be responsible for activating thiocar-
bamide prodrugs [26] and mutations in this gene make M.
tuberculosis drug-resistant. Heterologous expression of
EtaA enabled us to demonstrate that it represents a bona
fide BVMO acting on a number of ketones [27]. Recent
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studies have shown that by designing drugs for upregula-
tion of the etaA gene, efficacy of antitubercular prodrugs
can be enhanced [28]. This indicates that BVMOs can be
highly interesting as (pro)drug target asthey areprevalent
in proteomes of pathogenic microbes while no BVMO
gene is present in the human genome.
Tapping bacterial genomes for BVMOs
In order to obtain a robust BVMO, we used the BVMO-
identifying sequence motif to identify and clone a gene
from the mesothermophilic actinomycete Thermobifida
fusca. The respective enzyme, phenylacetone monooxy-
genase (PAMO), could be easily expressed in Escherichia
coli and was shown to be primarily active with aromatic
compounds [29]. Recent studies have shown that the
enzyme can be effectively used for regioselective and
enantioselective conversions of aromatic compounds [30–
32]. Another attractive feature of PAMO is its stability
toward higher temperatures and a range of solvents. In
fact, the use of solvents can also improve the enantiose-
lective behavior [33].
The direct harvesting of novel BVMOs by exploring
sequenced genomes has been intensified by the Grogan
group. Using effective ligation-independent cloning
(LIC) they have reported on cloning of 29 Type I BVMO
genes from the actinomycetes M. tuberculosis [34] and
Rhodococcus jostii [35]. This resulted in soluble expression
of most of the corresponding enzymes and for many of
them BVMO activity could be demonstrated. Clearly, the
approach of sifting through sequenced genomes for novel
BVMOs is productive.
Structural basis for biocatalytic Baeyer–
Villiger oxidations
The sudden increase in the discovery and characteriz-
ation of BVMOs eventually led to the elucidation of the
first BVMO crystal structure. Despite various unsuccess-
ful efforts with other BVMOs in the past, Mattevi and
coworkers managed to solve the crystal structure of
PAMO [36??]. The structure revealed that the monooxy-
genase exhibits a two-domain architecture with FAD-
binding and NADPH-binding domains, in which the
active site can be found in a cleft at the domain interface
(Figure 3). Remarkably, the conserved BVMO sequence
motif was found to be far from the active site, forming a
surface loop that connects the FAD and NADP domains.
The active site contains a strictly conserved arginine
(R337 in PAMO) which was suggested to play a role in
thestabilization ofthe
(Figure 2B). However, a recent detailed kinetic study
showed that this intermediate is still formed and stabil-
ized upon replacing Arg337 by an alanine [37?]. The
arginine appears crucial for facilitating the Baeyer–Villi-
ger reaction, most likely by (in)direct stabilization of the
Criegee-intermediate. The kinetic and structural data
suggested that a major conformational change occurs
during catalysis. Unfortunately, so far only one crystal
structure of PAMO has been obtained, which does not
contain a substrate or product, and thereby provides only
a structural snapshot. Recently, two NADP-complexed
crystal structures of a novel CHMO were solved which
revealed two different conformational states [38??].
These findings clearly confirm the structural dynamics
thatwaspredictedtooccurduringcatalysis.Theobserved
peroxyflavin intermediate
Baeyer–Villiger monooxygenases Torres Pazmin ˜o, Dudek and Fraaije139
Figure 1
Historical overview of progress in BVMO-related biocatalytic research.
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‘sliding’ ofthecoenzymeappearstoinvolvemultipleloop
movements, and includes repositioning of the conserved
active site arginine (Figure 2). These movements are
suggested to be coordinated by the previously mentioned
BVMO-motif, providing an explanation for the conserva-
tion of this sequence motif. However, more structural and
kineticinformation isstill needed inordertocomprehend
the way BVMOs are able to react with molecular oxygen,
activate the substrates and stabilize key reaction inter-
mediates. A key question concerning the catalytic mech-
anism of BVMOs remains: how do BVMOs efficiently
form andstabilize the
(Figure 2B)? Recent molecular dynamic simulation stu-
dies on other flavin-dependent oxidative enzymes have
provided the first insights into the mechanism that such
enzymesemploy toguide oxygen diffusiontothe reactive
moiety of the flavin cofactor [39]. However, the sub-
sequent steps in catalysis, oxygenation of reduced FAD
and stabilization of the peroxyflavin, remain mysterious.
peroxyflavinintermediate
While most of the reported BVMOs are sequence related,
some discovered BVMOs do not resemble these so-called
Type I BVMOs. One of these atypical BVMOs is
MtmOIV which represents a new type of FAD-depend-
ent and NADPH-dependent BVMOs that acts as key
enzyme in the biosynthetic pathway of mithramycin, a
polyketide anticancer agent. The recent elucidation of its
crystal structure and structure-based mutagenesis studies
have identified several residues that are involved in
substrate binding, although the crystal structures did
not reveal the exact binding of either NADP(H) or
mithramycin [40]. Unfortunately, MtmOIV displays a
narrow substrate specificity and hence appears less
relevant for biocatalysis.
Substrate acceptance plasticity of BVMOs
An inventory of the substrate acceptance profiles of the
newly cloned and studied BVMOs has revealed that each
BVMO has a preference for a certain class of compounds.
A BVMO substrate class can typically only be roughly
defined, by size and type of compounds. For example,
PAMO has evolved to convert a large set of aromatic
compounds while it hardly accepts aliphatic compounds
[29]. CHMO readily accepts a wide range of bulky cyclic
140
Biocatalysis and Biotransformation
Figure 2
(A) Some newly identified BVMO substrates: acetone, cyclopentadecanone, androstenedione (steroid), 1-deoxy-11-oxopentalenic acid
(sesquiterpenoid precursor), and hydroxyversicolorone (aflatoxin precursor). The site of oxygen insertion is indicated with an arrow. (B) A schematic
view of the BVMO mechanism as elucidated for CHMO and PAMO. The peroxyflavin intermediate is shown in red, reduced flavin in green, and oxidized
flavin in blue.
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aliphatic ketones but will not convert aromatic or simple
aliphatic compounds [5?]. The restriction in substrate
scope is still not well understood. For example, the
PAMO protein sequence is very similar to that of steroid
monooxygenase (STMO) (>50% seq. ident.) while it is
not active on steroids. Comparison of the crystal structure
of PAMO and a structural model of STMO does also not
provide clear clues on which residues are responsible for
the substrate discrimination. The ability of BVMOs to
adapt in such an apparently subtle way to different
substrateclasses hasprobablyresultedinwidelydisparate
roles in metabolic pathways. BVMOs are used to degrade
relatively small compounds, for example, an acetone-
specific BVMO has been found in a Gordonia species
[17], while other BVMOs act on highly complex biomo-
lecules such as aflatoxin derivatives [25]. Strikingly, no
BVMO has yet been identified that exploits in vivo the
ability of BVMOs to perform other types of oxidations,
such as a sulfoxidation reaction.
Redesign of BVMO for biocatalytic
applications
Despite the lacking of a crystal structure with bound
substrates, various attempts have been made to randomly
and rationally redesign BVMOs. Several studies have
been reported in which error-prone PCR was used to
generate diversity. Directed evolution of BmoF1 yielded
various mutants with an enhanced enantioselectivity
toward long-chain ketones [41?,42]. On the basis of a
homology model, these altered residues appear to be far
from the proposed active site. Also CHMO and CPMO
have been targeted by (semi-)random mutagenesis, yield-
ing several mutants with improved catalytic properties
[43–45]. Some of the uncovered mutation sites appear to
be in or near the active site. A more directed approach of
enzyme redesign was performed on the thermostable
PAMO. As this enzyme mainly acts on aromatic sub-
strates, it has been attempted to broaden its substrate
specificity to bulky, non-aromatic substrates. In the first
Baeyer–Villiger monooxygenases Torres Pazmin ˜o, Dudek and Fraaije141
Figure 3
Crystal structure of PAMO with the FAD cofactor and Arg337 highlighted in sticks. The FAD-binding domain is in green, the NADPH domain is in blue,
the Arg337 is shown in two conformations as observed in the crystal structure. The insets show details of the active sites of (1) PAMO, (2) CHMO in the
closed form, and (3) CHMO in the open form. Some key residues and the bulge are indicated.
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redesign study, an extended bulge was identified in the
structureofPAMO,whichisabsentinCHMO(seeinsets,
Figure 3). Removal of two residues in this loop region
yielded a variant that accepts the bulkier 2-phenylcyclo-
hexanone as a substrate [46]. A more extended mutagen-
esis study of the bulge region (residues 441–444 in
PAMO) yielded mutants that were able to enantioselec-
tively convert 2-phenylcyclohexanone and a derivative
[47]. Alternatively, mutation of Met446 to a glycine
resulted in a mutant enzyme with the altered substrate
specificity (e.g. formation of indigo blue from indole) and
displaying improved enantioselective behavior and
altered regioselectivity[32,48].Inarecentandinteresting
redesign studybytheReetzgroup twoconservedprolines
in the bulge region were targeted [49?]. Somewhat unex-
pectedly,thisrevealedthatreplacementofPro440renders
‘CHMO-like’mutants that areableto(enantioselectively)
convert 2-substituted cyclohexanone derivatives. Taken
together, the PAMO redesign studies indicate that one
loop region can play a dominant role in substrate discrimi-
nation. Furthermore, it is worth noting that all reported
PAMO mutants were shown to be thermostable which
underlines that this specific BVMO and its derivatives are
ideally suited for biocatalytic applications.
BVMO-based biocatalytic applications: shine
a light on BVMOs
As BVMOs require an electron donor (typically NADPH)
foractivity,BVMO-basedbiocatalyticapplications have to
deal with this in a cost-effective manner. In numerous
studies ithasbeen shown for recombinantcells expressing
BVMOsthatNADPHcanberegeneratedbymakinguseof
the coenzyme recycling system of the host (for recent
examples see [50–56]). In combination with in situ sub-
strate feeding and product removal, biotransformations
yielding up to 1 kg of Baeyer–Villiger product can be
performed [57?]. Successful attempts to improve the effi-
ciencyoftheB–Vreactioninrecombinantcellsincludethe
coexpression of glucose-6-phosphate dehydrogenase in E.
coli [58] and application of Corynebacterium glutamicum as
expression host [59]. The latter organism has been shown
to posses a high coenzyme regeneration capacity.
Recently, we have shown that by fusion engineering the
catalytic efficiency of E. coli expressing CHMO and
CPMO could be increased. In addition, by fusing several
BVMOs to phosphite dehydrogenase (PTDH), effective
Baeyer–Villiger oxidations could be performed using cell-
free systems [60?]. The efficiency of the self-sufficient
BVMO system was further improved by first, the addition
of an affinity purification tag; second, substitution of the
dehydrogenase subunit by a more thermostable PTDH;
and third, expansion to other members of the Type I
BVMO family [61].
Another elegant approach to circumvent the need for
coenzyme regeneration is the application of (sun) light
and a suitable sacrificial electron donor [62??]. The work
of Hollmann and coworkers has shown that light, with the
help of effective mediators, can be used to reduce the
flavin cofactor in BVMOs. This is a very attractive
approach albeit the reported efficiency is very poor.
Drastic improvements on this system are needed to
enable effective light-driven BVMO-mediated biocataly-
sis [63].
Conclusions
The last few years have seen a large and interesting
developmentofBVMO-related
(Figure 1). This has revealed that BVMOs are abundantly
present in nature and are responsible for widely varying
tasks. The biodiversity has not only been exploited by
classical cloning strategies, but also genome mining
efforts have led to a formidable increase in available
BVMOs. Biocatalytic studies have revealed that BVMOs
can be utilized for a startling number of different sub-
strates, while often exhibiting exquisite regioselectivity
and/or enantioselectivity.
through is the elucidation of BVMO structures which
has led to a better understanding of how these enzymes
function. However, many details on how separate cata-
lytic events take place remain unclear: for example, how
do BVMOs stabilize the peroxyflavin intermediate? And
what conformational changes occur during a full catalytic
cycle? Also a good view on the substrate binding pocket is
lacking and clearly asks for more structural studies on this
class of oxidative enzymes. This would enable more
directed enzyme redesign approaches for fine-tuning
BVMOs toward biocatalytic applications. Except for
the discovery or redesign of suitable BVMOs, challenges
lay ahead to develop cost-effective technical approaches
to apply BVMOs which should enable effective oxygen
supply and coenzyme recycling.
research studies
Anotherscientificbreak-
Acknowledgement
Research work of the authors is supported by the EU-FP7 OXYGREEN
research grant.
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The kinetic mechanism of wild-type and R337A PAMO was studied with
rapid kinetic analysis.
38.
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?
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?
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62.
??
This study shows that BVMOs can also be driven by light which opens
new avenues for developing coenzyme-independent applications of
redox biocatalysts.
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Biocatalysis and Biotransformation
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