8.

Baeyer-Villiger monooxygenases as useful biocatalysts

Marco W. Fraaije & Dick B. Janssen
Biotransformation and Biocatalysis Group
Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen

Biological Baeyer-Villiger oxidation

Baeyer-Villiger monooxygenases are enzyme that catalyze the insertion of an oxygen
atom in a ketone, next to the carbonyl carbon atom. This reaction converts a ketone into
an ester, a conversion that is of great value for the synthesis of interesting fine chemicals.
So far, only a limited number of Baeyer-Villiger monooxygenases have been identified
from bacteria and fungi. These enzymes typically contain FAD or FMN as a cofactor and
catalyze highly regio- and stereoselective oxygenations at the expense of NAD(P)H and
molecular oxygen.
Chemical routes for selective oxidations usually are catalyzed by heavy metals.
Enzymatic routes have been explored, but mainly for iron- or heme dependent enzymes
such as cytochrome P450. Flavin-dependent oxidases and monooxygenases represent a
relatively unexplored and attractive alternative group of enzymes, as they are able to
catalyze a huge variety of oxidation reactions while exhibiting a remarkable selectivity.
Our research focuses on discovery and exploration of novel flavin-containing Baeyer-
Villiger monooxygenases and alcohol oxidases. Besides exploring the biocatalytic
potential of these enzymes, we are also seeking a thorough understanding of their
catalytic and structural properties.

NADPH NADP+ O
O + H+ + O 2 + H2O
O
CHMO
Examples of Baeyer-Villiger monooxygenases.
O NADPH NADP+ CHMO, the classical cyclohexane
+ H+ + O 2 + H2O
O monooxygenase from Pseudomonas; HAPMO,
O
hydroxyacetophenone monooxygenase from
HO HAPMO HO Pseudomonas ACB; PAMO, phenylacetone
monooxygenase.
NADPH NADP+
+ H+ + O 2 + H2O
O

O
PAMO
O

The 4-hydroxyacetophenone monooxygenase from Pseudomonas ACB

Pseudomonas fluorescens strain ACB is able to degrade acetophenones by the initial
action of a Baeyer-Villiger monooxygenase. The enzyme converts acetophenones into
valuable aromatic esters which are synthons for the production of fine chemicals and
neuroactive pharmaceuticals. To obtain large amounts of this enzyme we have cloned the
gene cluster harboring the 4-hydroxyacetophenone monooxygenase. Large quantities of
the enzyme have been obtained after expression in E. coli using a modified pBAD vector.
This enabled us to explore the biocatalytic potential of this novel flavoprotein.

Table 2. Conversions catalyzed by EtaA from Mycobacterium tuberculosis.

N N O
ethionamide S S

NH2 NH2

methyl-p-tolylsulfide -
S S
O

O
phenylacetone
O

O
benzylacetone
O
O

bicyclohept-2-en-6-one O

O
O

2-hexanone O

O O

Sequence motifs for identifying BVMOs

We have recently described a protein sequence motif (FXGXXXHXXXW[DP]) that
can be used to identify Baeyer-Villiger monooxygenases (FRaaije et al., 2002). Site-
directed mutagenesis using 4-hydroxyacetophenone monooxygenase as a model has
shown that conserved residues in this motif are of crucial importance for BVMO activity.
All recombinant Baeyer-Villiger monooxygenases characterized so far contain this
protein sequence motif. In order to find novel Baeyer-Villiger monooxygenases that can
be of use for biocatalysis we have screened all available bacterial genomes using the
Baeyer-Villiger monooxygenase-specific sequence motif. It was found that Baeyer-
Villiger monooxygenases could be identified in a large number of genomes. While many
genomes seem to be devoid of these monooxygenases, several bacteria and fungi contain
a multitude of Baeyer-Villiger monooxygenases. In total, more than 100 putative Baeyer-
Villiger monooxygenase sequences can be retrieved from the current microbial genome
database using the BVMO motif. This numbers sharply contrasts with the number of
available recombinant Baeyer-Villiger monooxygenases.
The EtaA flavin monooxygenase of Mycobacterium tuberculosis is a Baeyer-
Villiger monooxygenase
Hinted by the presence of this motif we could demonstrate that the etaA gene, that is
held responsible for prodrug activation in Mycobacterium tuberculosis, also represents a
Baeyer-Villiger monooxygenase. We have overexpressed this NADPH-dependent
mycobacterial monooxygenase in Escherichia coli. The purified enzyme was shown to
perform Baeyer-Villiger reactions with a remarkably broad range of carbonylic substrates
(Fraaije et al., 2004). Except for Baeyer-Villiger reactions, it is also able to catalyze
(enantioselective) sulfoxidation reactions. Sulfoxidation of ethionamide forms the
molecular bases of the prodrug activating activity of EtaA.

The first structure of a Baeyer-Villiger monooxygenase

Two putative Baeyer-Villiger monooxygenase genes were found in the genome of
Thermobifida fusca, a moderately thermophilic soil bacterium that typically grows at
55˚C. We have overexpressed one of these Baeyer-Villiger monooxygenases in E. coli.
The purified enzyme was indeed shown to be active as a Baeyer-Villiger monooxygenase
acting on a wide range of aromatic and aliphatic ketones. The best substrate found so far
is phenylacetone. As a consequence the enzyme is named phenylacetone monooxygenase
(PAMO). Although PAMO shows a relatively high sequence identity (53 %) with steroid
monooxygenase, no activity could be observed with progesterone. This illustrates that it
is difficult to predict substrate specificity of a certain enzyme based on its sequence.

The first crystal structure of a Baeyer-Villiger monooxygenase: phenylacetone

monooxygenase.

In collaboration with the group of Prof. Dr. A. Mattevi (Pavia, Italy) we have recently
solved the structure of this thermostable Baeyer-Villiger monooxygenase by X-ray
crystallography, representing the first crystal structure of a Baeyer-Villiger
monooxygenase (Malito et al., 2004a). As PAMO shows significant sequence homology
to known Baeyer-Villiger monooxygenases, e.g. cyclohexanone monooxygenase, this
structure can be exploited as prototype structure for future studies.

Further reading
Fraaije MW, Kamerbeek NM, van Berkel WJ, Janssen DB. 2002. Identification of a Baeyer-
Villiger monooxygenase sequence motif. FEBS Lett. 518:43-7.
Kamerbeek NM, Olsthoorn AJ, Fraaije MW, Janssen DB. 2003. Substrate specificity and
enantioselectivity of 4-hydroxyacetophenone monooxygenase. Appl Environ Microbiol 69:419-
26.
Malito E, Alfieri A, Fraaije MW, Mattevi A. 2004a. Crystal structure of a Baeyer-Villiger
monooxygenase. Proc Natl Acad Sci U S A 101:13157-62.
Malito E, Coda A, Bilyeu KD, Fraaije MW, Mattevi A. 2004b. Structures of Michaelis and product
complexes of plant cytokinin dehydrogenase: implications for flavoenzyme catalysis. J Mol Biol.
341:1237-49.
Kamerbeek NM, Fraaije MW, Janssen DB. 2004. Identifying determinants of NADPH specificity
in Baeyer-Villiger monooxygenases. Eur J Biochem. 271:2107-16.
Fraaije MW, Kamerbeek NM, Heidekamp AJ, Fortin R, Janssen DB. 2004. The prodrug activator
EtaA from Mycobacterium tuberculosis is a Baeyer-Villiger monooxygenase. J Biol Chem.
279:3354-60.
Fraaije MW, Wu J, Heuts DP, van Hellemond EW, Spelberg JH, Janssen DB. 2005. Discovery of
a thermostable Baeyer-Villiger monooxygenase by genome mining. Appl Microbiol Biotechnol
66:393-400.