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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.

HO

NADPH + H + + O 2 NADP + + H 2 O O O O
NADPH
+ H + + O 2
NADP +
+ H 2 O
O
O
O
CHMO
O
NADPH
+ H + + O 2
NADP +
+ H 2 O
O
O
HAPMO
HO
NADPH
+ H + + O 2
NADP +
+ H 2 O
O
O
PAMO

O

Examples of Baeyer-Villiger monooxygenases.

CHMO, the classical cyclohexane

monooxygenase from Pseudomonas; HAPMO,

hydroxyacetophenone monooxygenase from

Pseudomonas ACB; PAMO, phenylacetone

monooxygenase.

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.

ethionamide

methyl-p-tolylsulfide

phenylacetone

benzylacetone

bicyclohept-2-en-6-one

2-hexanone

N S NH 2
N
S
NH 2
N O S NH 2
N
O
S
NH 2
S O
S
O
- S O
-
S
O
  • O

O

the enzyme have been obtained after expression in E. coli using a modified pBAD vector. This
  • O

O

O O
O
O
O O
O
O
the enzyme have been obtained after expression in E. coli using a modified pBAD vector. This

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 EtaA flavin monooxygenase of Mycobacterium tuberculosis is a Baeyer- Villiger monooxygenase Hinted by the presence

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.