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Biochemical properties and catalytic scope of a Baeyer-Villiger monooxygenaseKamerbeek, Nanne

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1 General introduction

Chapter 1

1. FLAVIN (BIO)CHEMISTRY The most versatile class of cofactors present in nature are the flavins (Massey, 2000). The yellow color of flavins [Latin, flavus: yellow] is caused by the flavin core component: the isoalloxazine ring in its oxidized form (Figure 1). Attachment of a ribose moiety to the N10 of the isoalloxazine ring yields riboflavin. Plants and microorganisms are able to synthesize riboflavin themselves while mammals need it as an ingredient in their diet. Riboflavin is also known as vitamin B2. To serve as a cofactor, riboflavin is phosphorylated by a flavokinase, yielding flavin mononucleotide (FMN). Conjugation of FMN with adenosine monophosphate (AMP), catalyzed by FAD synthetase, yields the most common flavin cofactor: flavin-adenine dinucleotide (FAD).

NN

N N

NH2

O

OH OHP OO

P OO

OO

CH2

NH

N NH

NH

O

OCH3

CH3

R

CH3

O

CH2

N

N N

NH

O

OCH3

CH3

( )3

85 4a 3

110 2H+, 2e-

riboflavin

FMN

FAD

HCOH

flavinred flavinox

Figure 1. Structural formulas of flavins. Enzymological research of the last decades has shown that flavin-containing enzymes can catalyze a wide range of biological redox reactions and they play a role in numerous biological degradation and synthesis pathways. Next to catalyzing redox reactions, flavoproteins also play a role in many other crucial biological processes, e.g. electron transport, light-dependent repair of DNA damage (Jorns et al., 1987), light sensing (Hsu et al., 1996; Sancar, 2000), and bioluminescence (Tu and Mager, 1995). The molecular basis for the functional adaptability of flavoproteins is based on a unique feature of the cofactor: the capability of a flavin molecule to exist in both a one-electron reduced state and a two-electron reduced state. As a consequence, flavoproteins can participate both in one-electron redox reactions (e.g. electron transport) and in reactions that involve a transfer of two electrons (e.g. oxidation reactions).

8

General introduction

2. FLAVOENZYMES 2.1 Classification At present several hundreds of flavoenzymes have been characterized and described in the literature. Most of these flavoenzymes contain a non-covalently bound flavin as prosthetic group, but there are some that contain covalently bound FAD or FMN (Mewies et al., 1998). For example, vanillyl-alcohol oxidase contains FAD linked to a histidine (de Jong et al., 1992), and p-cresol methyl hydroxylase contains FAD linked to a tyrosine (McIntire et al., 1985). In case of vanillyl-alcohol oxidase it has been shown that such a covalent linkage is beneficial for catalysis as it enhances the oxidative power of the flavin cofactor (Fraaije et al., 1999). Classification of flavoenzymes has been done using different criteria, such as the type of chemical reaction that is catalyzed, the nature of the reducing and oxidizing substrates, and the topology of their 3-D structures (Massey, 2000). In a recent review, Palfey and Massey used a classification based on the first criterion (Palfey and Massey, 1996). This classification was used for flavoproteins in which the flavin is directly involved in a catalytic event and it is summarized in a slightly modified form in Table 1. A short description of each class is given below. 2.2 Oxidases Oxidases catalyze the 2-electron oxidation of organic molecules, followed by oxidation of the flavin by O2. During the reductive half-reaction of flavoprotein oxidases, the flavin is reduced by two electrons originating from a C-H or C-heteroatom bond. Although the exact molecular mechanism of flavin reduction has been debated for several decades, it appears that in case of alcohol oxidation, the electrons are transferred to the flavin via a direct transfer of a hydride (Fraaije and Mattevi, 2000; Sobrado and Fitzpatrick, 2003). For the oxidation of amines, a radical mechanism has been proposed in which the flavin is reduced in two one-electron reduction steps (Edmondson, 1995). The common denominator for oxidases is the fact that in the subsequent oxidative half-reaction, the reduced flavin transfers the electrons to molecular oxygen yielding hydrogen peroxide. 2.3 Dehydrogenases/reductases Like oxidases, dehydrogenases and reductases are able to transfer electrons from one substrate to another. However, this class of enzymes is unreactive with molecular oxygen. Instead, dehydrogenases oxidize an organic compound with the use of a specific electron acceptor (e.g. NADP+) while reductases transfer electrons from a specific electron donor (e.g. NADH) to their organic substrate. A well-studied class of flavin-containing dehydrogenases are the acyl-CoA dehydrogenases (Ghisla and Thorpe, 2004). These enzymes have a similar catalytic mechanism as several oxidases as substrate oxidation proceeds via abstraction of a hydride. The reduced flavin will donate these electrons in a subsequent redox reaction to a suitable electron acceptor. An example of a flavin-containing reductase is ferredoxin-NADP+ reductase. This FAD-containing enzyme plays a role in photosynthesis. It reduces NADP+ with ferredoxin as electron donor.

9

10

Chapter 1

Table 1. Classification of flavoenzymes with some representative examples. S = substrate, P = product, NH = e- donor, N+ = e- acceptor. Enzyme class

Example

E.C.

number

Ref.

Oxidases S + O2 P + H2O2

Glucose oxidase Vanillyl-alcohol oxidase Monoamine oxidase D-amino acid oxidase

1.1.3.4 1.1.3.38 1.4.3.4 1.4.3.3

(Hecht et al., 1993) (de Jong et al., 1992) (Binda et al., 2002)

(Ronchi et al., 1982) Dehydrogenases/Reductases S + N+ P + NH S + NH P + N+

Acyl-CoA dehydrogenase D-lactate dehydrogenase Ferredoxin-NADP+ reductase Cytochrome P450 reductase

1.3.99.3 1.1.1.28 1.18.1.2 1.6.2.4

(Ghisla and Thorpe, 2004) (Garvie, 1980)

(Karplus et al., 1991) (Hubbard et al., 2001)

Disulfide oxidoreductases S + NH P + N+

Glutathione reductase Thioredoxin reductase Lipoamide dehydrogenase Mercuric reductase

1.6.4.2 1.6.4.5 1.8.1.4 1.16.1.1

(Pai and Schulz, 1983) (Waksman et al., 1994)

(Mattevi et al., 1992) (Schiering et al., 1991)

Monooxygenases external S + O2 + NH P + H2O + N+ internal S + O2 P + H2O

p-Hydroxybenzoate hydroxylase Phenol hydroxylase Cyclohexanone monooxygenase Lactate 2-monooxygenase

1.14.13.2 1.14.13.7 1.14.13.22 1.13.12.4

(Entsch and van Berkel, 1995)

(Neujahr and Gaal, 1973) (Donoghue et al., 1976)

(Sullivan, 1968)

H+ H+

H+

H+

H+

G

eneral introduction

Table 1. continued. Enzyme class

Example

E.C.

number

Ref.

Non-redox flavoenzymes S P

Alkyl-dihydroxyacetonephosphate synthase UDP-galactopyranose mutase Hydroxynitrile lyase

2.5.1.26 5.4.99.9 4.1.2.1.10

(de Vet et al., 2000)

(Fullerton et al., 2003) (Dreveny et al., 2002)

11

Chapter 1

Another well-known example of a flavin-containing reductase is cytochrome-P450 reductase. Many biological oxygenations are catalyzed by cytochrome P450s. These heme-containing enzymes activate oxygen and for this electrons are needed. The required electrons are often provided by a flavin-containing cytochrome-P450 reductase that contains both FAD and FMN and reduces cytochrome-P450 at the expense of NADPH (Hubbard et al., 2001). A special case is the bacterial cytochrome-P450 BM-3 from Bacillus megaterium, as this enzyme combines both the reductase and cytochrome in one polypeptide (Narhi and Fulco, 1987). 2.4 Disulfide oxidoreductases A specific class of flavoenzymes uses both an FAD cofactor and cysteine residues as electron transfer mediators. These flavoprotein disulfide oxidoreductases are important for cellular energy metabolism and play a role in protection from damage by molecular oxygen and toxic compounds. During the reaction cycle, NAD(P)H reduces the flavin which subsequently reduces an active site disulfide. The cysteines undergo thiol-disulfide interchange with a disulfide substrate, like glutathione or thioredoxin (Kuriyan et al., 1991). Thioredoxin reductases from different organisms, including bacteria, the malaria parasite Plasmodium falciparum, and the human enzyme, show a surprising diversity in mechanism. This is the basis for efforts to develop thioredoxin reductase inhibitors that can be used to treat a variety of microbial infections and parasitic diseases (Becker et al., 2000). 2.5 Monooxygenases Like oxidases, flavoprotein monooxygenases also react with oxygen but for a different purpose. Monooxygenases insert an oxygen atom derived from molecular oxygen in an organic substrate. For such a monooxygenation reaction, monooxygenases require, besides O2, supply of electrons. Internal flavoprotein monooxygenases obtain these reducing equivalents from the substrate (XH2) itself (see scheme below). One example of an internal monooxygenase is lactate monooxygenase, an enzyme that catalyzes the oxidative decarboxylation of lactate to acetate, carbon dioxide and water (Sullivan, 1968). However, most flavoprotein monooxygenases represent external flavoprotein monooxygenases. External flavoprotein monooxygenases receive the reducing equivalents from external donors, such as NADPH or NADH. Internal monooxygenase reaction: XH2 + O2 → XO + H2O External monooxygenase reaction: X + O2 + DH + H+ → XO + H2O + D+

Scheme 1. General reaction schemes of flavoprotein monooxygenases 2.6 Non-redox flavoenzymes Several flavoproteins have been found to catalyze non-redox reactions. For example, alkyl-dihydroxyacetonephosphate synthase uses its flavin as an electron sink during a transferase-type of reaction (de Vet et al., 2000). Also for the recently studied UDP-galactopyranose mutase, the flavin is likely to participate in catalysis since a reduced form of the cofactor seems to be crucial for

12

General introduction

activity (Soltero-Higgin et al., 2004). The recent structure determination of FAD-containing hydroxynitrile lyase from almond (Prunus amygdalus) revealed that, although the flavin is near the active site, it is too far from the substrate to promote redox catalysis. In this case, the oxidized flavin assists in catalysis by creating the required electrostatic environment while it also may have a structural role (Dreveny et al., 2002). 3. FLAVOPROTEIN MONOOXYGENASES The concerted reaction between O2 and carbon in organic compounds is spin-forbidden. Nevertheless, a large number of enzymes have found a way to use molecular oxygen as a substrate. For such reactivity an enzyme has to be able to activate molecular oxygen. To create a species that transfers molecular oxygen, enzymes often use a transition metal, which may or may not be bound to an organic cofactor like pteridin or heme. Alternatively, reduced flavin is able to use molecular oxygen as a substrate without the help of any metal (Massey, 1994). Upon one electron-transfer from reduced flavin to oxygen, a complex is formed of O2

- and the flavin radical. A subsequent spin inversion results in formation of reduced oxygen (Ghisla and Massey, 1989). In case of an oxidase, hydrogen peroxide is formed. For most flavoprotein monooxygenases, a covalent adduct between the C(4a) of the flavin and molecular oxygen is formed and stabilized, yielding a reactive C(4a)-hydroperoxyflavin species (Figure 2).

NH

N N

NH

O

OCH3

CH3

R

R

N

N N

NH

O

OCH3

CH3

NH

N N

NH

O

OCH3

CH3

R

OO

NH

N N

NH

O

OCH3

CH3

R

OOH

NH

N N

NH

O

OCH3

CH3

R

OH

+ H+

- H+

XO

X

XO

X, H+

electrophilic oxygenation

nucleophilic oxygenation

hydroflavin oxidized flavin

reduced flavin hydroperoxyflavin peroxyflavin

O2

NAD(P)+, H2O

NAD(P)H

Figure 2. General mechanism of oxygenation reactions catalyzed by external flavoprotein monooxygenases.

13

Chapter 1

In solution such a peroxyflavin is unstable and decays to hydrogen peroxide and oxidized flavin. However, flavoprotein monooxygenases stabilize this species in such a way that it can oxygenate a substrate (Entsch and van Berkel, 1995). Depending on the protonation state of the peroxyflavin, it can perform a nucleophilic or an electrophilic attack on the substrate. As a result, a single atom of molecular oxygen is incorporated into the substrate, while the other oxygen atom is reduced to water (Figure 2).

4. CLASSIFICATION OF EXTERNAL FLAVOPROTEIN MONOOXYGENASES Most flavoprotein monooxygenases that are known are external monooxygenases and can be grouped in three classes on basis of protein sequence, biochemical properties and catalytic mechanism: class A, B and C. Besides this, Discrimination is based on mechanistic differences and specific structural features which are reflected in sequence similarity (protein sequence motifs). The typifying characteristics of each class are indicated below, while the next paragraphs provide a more elaborate description of members belonging to each class. Examples can be found in Table 2.

Class A

• encoded by a single gene • typically contain a tightly bound FAD cofactor • specifically depend on NADPH as coenzyme • are structurally composed of two dinucleotide binding domains (Rossmann fold) binding FAD

and NADPH, respectively • keep the coenzyme NADPH/NADP+ bound during catalysis

Class B

• encoded by a single gene • typically contain a tightly bound FAD cofactor • depend on NADPH or NADH as coenzyme • contain one dinucleotide binding domain (Rossmann fold) binding FAD • bind the coenzyme NAD(P)H only during the reductive half-reaction

Class C

• encoded by at least two genes • use FAD or FMN as a coenzyme • can use NADPH and/or NADH as coenzyme • reduced flavin coenzyme is often transferred between subunits

14

Table 2. Examples of external flavoprotein monooxygenases

Class

example

subunit size

(kDa)

structure

origin

references

A B C

4-hydroxyacetophenone monooxygenase cyclohexanone monooxygenase flavin-containing monooxygenase 3 lysine N6-hydroxylase p-hydroxybenzoate hydroxylase salicylate hydroxylase phenol hydroxylase biphenyl 3-monooxygenase 4-hydroxyphenylacetate 3-monooxygenase styrene monooxygenase 2,5-diketocamphane 1,2-monooxygenase

72 61 65 50

44 47 76 60

α = 59 β = 19

α = 43 β = 18

α = 39 β = 32

α2 α α2 α4

α2 α2 α2 α4

αβ

αβ

α2β

Pseudomonas fluorescens Acinetobacter NCIMB 9871 Homo sapiens Escherichia coli Pseudomonas fluorescens Pseudomonas putida Trichosporon cutaneum Pseudomonas azelaica Escherichia coli Pseudomonas VLB120 Pseudomonas putida

(Kamerbeek et al., 2001) (Donoghue et al., 1976) (Ziegler, 2002) (Plattner et al., 1989) (Entsch and van Berkel, 1995) (White-Stevens and Kamin, 1972) (Neujahr and Gaal, 1973) (Suske et al., 1997) (Prieto and Garcia, 1994) (Otto et al., 2004) (Jones et al., 1993)

General introduction

15

Chapter 1

4.1 Class A flavoprotein monooxygenases This class is also referred to as multifunctional flavin-containing monooxygenases as representatives of this class are able to oxidize both carbon atoms and other atoms. This class comprises three sequence-related flavoprotein monooxygenase families: the so-called flavin-containing monooxygenases (FMOs), the microbial N-hydroxylating monooxygenases (NMOs) and the Type I Baeyer-Villiger monooxygenases (BVMOs). All members of these three monooxygenase families are single-component FAD-containing enzymes and specific for NADPH. The proteins sequences of these monooxygenases contain two Rossmann-fold motifs indicative for two binding domains for FAD and NADPH. FMOs were originally identified in liver microsomes and named ‘mixed-function oxidases’. The name of these enzymes was later changed to flavin-containing monooxygenase (FMO) (Ziegler, 2002). FMOs are found in all mammals and other eukaryotic organisms. In mammals and human several isoforms exist, each having a different substrate specificity and tissue localization. Six FMO genes and five FMO pseudogenes have been identified in the human genome. FMO3 is the most important isoform in the liver (Furnes et al., 2003; Hernandez et al., 2004). FMOs play an important role in detoxification of drugs and other xenobiotics in the human body thereby complementing the activities of the cytochrome P450 system. The membrane-associated enzymes catalyze the monooxygenation of carbon-bound reactive heteroatoms: nitrogen, sulfur, phosphorus, selenium, or iodine. In contrast to mammalian FMOs, yeast FMO does not oxidize nitrogen-containing compounds but is only active with biological thiols (Suh et al., 1996). It was shown that the yeast enzyme is required for proper folding of disulfide-containing proteins by generating an oxidizing environment in the endoplasmatic reticulum (Suh et al., 1999). Microbial NMOs, catalyzing the N-hydroxylation of long-chain primary amines, play a role in the biosynthesis of various bacterial and fungal siderophores (low molecular weight iron chelators) (Stehr et al., 1998). Cloning and sequencing of several NMO genes has revealed that they share sequence homology with FMOs (Chapter 3). As for FMOs, NMOs need NADPH to perform catalysis. Limited biochemical data are available for these enzymes, which is partly caused by their low affinity for FAD hindering mechanistic studies (Stehr et al., 1999). Type I BVMOs are also sequence related to FMOs and NMOs (Chapter 3). Type I Baeyer-Villiger monooxygenases are discussed in more detail in section 5. 4.2 Class B flavoprotein monooxygenases Members of this group of enzymes are usually involved in the initial microbial degradation of aromatic compounds by hydroxylation of the aromatic ring (Moonen et al., 2002). The prototype enzyme of this class is p-hydroxybenzoate hydroxylase (PHBH) from Pseudomonas that has been studied comprehensively (Entsch and van Berkel, 1995). Other members are 2-hydroxybiphenyl 3-monooxygenase from Pseudomonas azelaica (Suske et al., 1997), phenol hydroxylase from Trichosporon cutaneum (Neujahr and Gaal, 1973), and salicylate hydroxylase from Pseudomonas putida, the latter being the first characterized enzyme of this class (White-Stevens and Kamin, 1972). Class B monooxygenases can be NADPH or NADH dependent. The C(4a)-hydroperoxide is the oxygenating flavin species, that performs an electrophilic attack on the aromatic ring (Figure 2).

16

General introduction

Typical substrates are aromatic compounds that already contain a hydroxyl group (activated ring). This is an important difference with the P450 enzymes that can also hydroxylate non-activated aliphatic or aromatic compounds. The 3-D structure has been solved for PHBH (Schreuder et al., 1989; Wierenga et al., 1979) and phenol hydroxylase from Trichosporon cutaneum (Enroth et al., 1998). Both enzymes are single polypeptides and their sequences contain two sequence motifs for the FAD binding region. The N-terminal GxGxxG sequence is indicative for the βαβ-fold (or Rossmann fold) that binds the ADP moiety of FAD (Wierenga et al., 1986). The amino acids of the second motif, GD, are in contact with the riboflavin moiety of FAD (Eggink et al., 1990). Interestingly, there is no distinct domain that binds the coenzyme NADPH. This is in line with the fact that NADPH only forms a transient complex to reduce the flavin cofactor. The formed NADP+ is rapidly released. Nevertheless, recognition of NADPH is very specific which has triggered several groups to study the structural basis of coenzyme recognition by PHBH. Eppink and colleagues have described an additional fingerprint sequence for this class of monooxygenases. The fingerprint, containing a highly conserved DG motif, appeared to be involved in both the binding of the pyrophosphate moiety of FAD and the recognition of the NADPH coenzyme (Eppink et al., 1997b). Furthermore, recent mutagenesis and crystallographic studies have revealed a plausible binding mode for NADPH in PHBH (Eppink et al., 1999; Wang et al., 2002). 4.3 Class C flavoprotein monooxygenases In addition to the single polypeptide flavoprotein monooxygenases discussed in the previous two sections, multi-component monooxygenases have been discovered that use flavin as a substrate rather than a cofactor. The variety in this group of monooxygenases is quite large concerning cofactor use, coenzyme specificity and sequence homology (Chaiyen et al., 2001). Generally, these enzymes consist of two different polypeptide chains that have different functions: a reductase component carrying out the flavin reduction and an oxygenase component oxidizing the substrate by molecular oxygen. The reducing equivalents needed for the oxygenation are transferred from NAD(P)H to a flavin that is bound to the reductase component. Subsequently, the reduced flavin is transferred to the oxygenase component. Upon reaction with molecular oxygen the monooxygenation reaction takes place. 4-Hydroxyphenylacetate-3-monooxygenase from Escherichia coli W is a prototype of this family and consists of the HpaB oxygenase (58.8 kDa) and the HpaC reductase (18.7 kDa) (Galan et al., 2000; Prieto and Garcia, 1994). The 4-hydroxyphenylacetate-3-hydroxylase from Pseudomonas putida catalyzes the same reaction but for this enzyme flavin reduction and substrate oxygenation take place in the same large component. In this case the second component is needed for productive hydroxylation (Arunachalam et al., 1994). For some enzymes, one flavin molecule is not sufficient for catalysis. For example, phenol hydroxylase from thermophile Bacillus thermoglucosidasius A7 consists of an oxygenase (PheA1) and a flavin reductase (PheA2) that uses NADH. PheA2 harbors as prosthetic group a tightly bound FAD that reduces free flavins (Kirchner et al., 2003). Thus, electrons are transferred from NADH via PheA2-bound FAD to exogenous free FAD, which is then transferred to PheA1 (van den Heuvel et al., 2004). Another example of a multi-component monooxygenase is styrene

17

Chapter 1

monooxygenase from Pseudomonas sp. VLB120 that consists of a reductase component, StyA, and a monooxygenase component, StyB (Otto et al., 2004). This enzyme was found to be of value for biotechnological applications as it catalyzes highly enantioselective epoxidation reactions (Schmid et al., 2001b). In addition to hydroxylation and epoxidation reactions, desulfurization reactions may also be carried out by two-component flavin-dependent monooxygenases. Dibenzothiophene desulfurization has been studied in a variety of microorganisms (Gray et al., 2003). The genes responsible for the degradation pathway have been cloned from Rhodococcus sp. IGTS8 (Denome et al., 1994; Matsubara et al., 2001; Piddington et al., 1995). The complete removal of sulfur from the substrate requires four enzymes. The DszA and DszC proteins are both FMN-dependent monooxygenases and require DszD, a flavin reductase, for activity. DszD uses NADH as electron source and is related to the reductases of the multi-component monooxygenases discussed above (HpaC, StyB and PheA2). These desulfurization reactions are particularly interesting for biotechnological applications and some aspects will be discussed further in the section on biocatalysis (§ 6). The fourth enzyme, DszB, is a desulfinase and catalyzes the last step, yielding 2-hydroxybiphenyl and SO3

2-. Some other multi-component flavin-dependent monooxygenases were shown to be involved in the synthesis of antibiotics in Streptomyces species (Kendrew et al., 1995; Parry and Li, 1997; Thibaut et al., 1995) and the microbial degradation of chelating compounds, illustrating their widespread occurrence (Uetz et al., 1992). 5. A SPECIAL CLASS OF MONOOXYGENASES: BAEYER-VILLIGER MONOOXYGENASES In 1899, Adolf von Baeyer and Victor Villiger published an article in which they described the discovery of a reaction that involved the conversion of ketones into esters or cyclic ketones into lactones (Baeyer and Villiger, 1899) (Figure 3). Since then, this so-called Baeyer-Villiger reaction has received much attention in organic chemistry (Renz and Meunier, 1999). Almost fifty years later, a biological Baeyer-Villiger reaction was reported to be involved in the biotransformation of steroids by fungi (Turfitt, 1948). From then on, a large number of Baeyer-Villiger oxidations were discovered to play a role in microbial biosynthetic and degradation pathways (Chapter 7) (Kamerbeek et al., 2003b). In some cases, this led to the characterization of the responsible enzymes; the Baeyer-Villiger monooxygenases (BVMOs). All BVMOs discovered so far depend on a flavin cofactor for catalysis. Based on mechanistic studies with cyclohexanone monooxygenase, it is assumed that the flavin cofactor in these BVMOs forms a so-called peroxyflavin intermediate that carries out the oxidation reaction (Sheng et al., 2001). The peroxyflavin intermediate in fact resembles the peracids that are used in organic chemistry to perform Baeyer-Villiger oxidations (Renz and Meunier, 1999). Based on biochemical data, Willetts has classified BVMOs in two types: Type I and Type II BVMOs (Willetts, 1997). Recent

18

General introduction

biochemical studies on these monooxygenases have revealed that Type I BVMOs belong to the class A flavoprotein monooxygenases, while Type II BVMOs belong to the class C flavoprotein monooxygenases.

peracid

ketone ‘Criegee’ intermediaterearrangement

OOHR3

O

O

OR1R2

OHR3

O

O

R2R1

O

R3

O

OHR2R1

O

+

ester or lactone acid Figure 3. Baeyer-Villiger oxidation reaction mechanism. 5.1 Type I BVMOs Type I BMVOs are composed of a single polypeptide and contain FAD as a cofactor while NADPH serves as electron donor. The properties of Type I BVMOs that have been cloned and characterized are reviewed in Chapter 7 of this thesis. The prototype of Type I BVMOs is cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIB 9871. It was originally isolated and characterized in 1976 by Trudgill and coworkers (Donoghue et al., 1976). Extensive research has shown that CHMO carries out Baeyer-Villiger oxidations on a wide variety of cyclic ketones with exquisite chemo-, regio-, and enantioselectivities (Mihovilovic et al., 2002b; Stewart, 1998b).The CHMO gene was cloned in 1988 (Chen et al., 1988) and 11 years later the next Type I BVMO, the steroid monooxygenase gene from Rhodococcus rhodochrous, was cloned (Morii et al., 1999). From then on more sequences became steadily available including the genes encoding 4-hydroxyacetophenone monooxygenase (Chapter 2), cyclododecanone monooxygenase (Kostichka et al., 2001), and cyclopentanone monooxygenase (Iwaki et al., 2002). Sequence similarity analysis has shown that Type I BVMOs can be identified using a specific protein sequence motif (Chapter 3). 5.2 Type II BVMOs Type II BVMOs are composed of several subunits and as a result they belong to class C flavoprotein monooxygenases (see § 4.3, Table 2). Only a few BVMOs belonging to this class of flavoprotein monooxygenase have been described in the literature (Taylor and Trudgill, 1986). The best described examples come from Pseudomonas putida ATCC 17453 (NCIMB 10007), a strain

19

Chapter 1

that is able to grow on camphor. To degrade this compound, the bacterium recruits three BVMOs (two of Type II and one of Type I). Initial degradation is catalyzed by the Type II BVMOs. The organism uses 2,5-diketocamphane 1,2-monooxygenase or 3,6-diketocamphane 1,6-monooxygenase for ring expansion of (+)- or (-)-camphor respectively. Taylor et al. presented a study in which they show that the 2,5-diketocamphane 1,2-monooxygenase is built up by an oxygenase component and an flavin reductase component (Taylor and Trudgill, 1986). The oxygenase component (78 kDa) consists of two subunits of equal size and strongly binds one FMN molecule. The small component is an NADH-dependent reductase (36 kDa), reducing the FMN bound to the oxygenase. Jones et al. characterized the 3,6-diketocamphane 1,6-monooxygenase from cells grown on (-)-camphor which revealed a close resemblance to 2,5-diketocamphane 1,2-monooxygenase (Jones et al., 1993). Interestingly, several bacterial luciferases have also been classified as Type II BVMOs (Villa and Willetts, 1997) as they display a similar oligomeric structure and catalyze a reaction similar to a Baeyer-Villiger oxidation (Eckstein et al., 1993). Consistent with this, it was shown that the luciferases from Vibrio fischeri and Photobacterium phosphoreum could perform Baeyer-Villiger reactions on different ketones (Villa and Willetts, 1997). Nevertheless, the above-mentioned Type II BVMOs have never been reported to emit light during catalysis. For both Type I and Type II BVMOs, no crystal structure exists. As a consequence, the molecular basis for the formation and the stabilization of the proposed peroxyflavin intermediate has remained unclear so far. 6. BIOCATALYSIS WITH FLAVIN-DEPENDENT MONOOXYGENASES Regio- and/or enantioselective insertion of oxygen is not straightforward to be accomplished when using chemical techniques. Therefore, flavin-dependent monooxygenases represent promising biocatalytic tools (Schmid et al., 2001a). This section will discuss some of the monooxygenases that have been shown to be of value for biocatalytic processes. 6.1 Styrene monooxygenase Enantiopure styrene oxides are important building blocks for the pharmaceutical industry. A promising example of an enantioselective monooxygenase is styrene monooxygenase from Pseudomonas sp. VLB120 (§ 4.3). This enzyme catalyzes the conversion of styrene into (S)-styrene oxide at an enantiomeric excess larger than 99% (Panke et al., 1998). A recombinant E. coli expressing StyA and StyB was developed (Panke et al., 2000) and this whole-cell biocatalyst showed a broad substrate spectrum that gives access to various chiral aryloxides (Schmid et al., 2001b). The system was successfully scaled-up and it was possible to produce almost 400 g (S)-styrene oxide at pilot-scale (30 L fed-batch bioconversion, two-liquid phase system) (Panke et al., 2002). Introduction of an apolar organic solvent reduces toxic effects of substrate and/or product and the use of whole cells instead of an isolated biocatalyst has the advantage of in vivo coenzyme regeneration. The same research group that developed this whole cell biocatalyst also designed a

20

General introduction

small-scale cell-free system. The only enzymatic component of this system is the monooxygenase (StyA). The reductase component (StyB), NADH and an NADH regenerating system could be replaced by the organometallic complex pentamethyl-cyclopentadienyl rhodium bipyridine [Cp*Rh(bpy)(H2O)]2+. This redox-catalyst receives electrons from the oxidation of formate to carbon dioxide and is able to regenerate reduced FAD directly. The epoxidation rate of this chemo-enzymatic system was ~ 70% of a fully enzymatic reaction (Hollmann et al., 2003). 6.2 Hydroxybiphenyl 3-monooxygenase Held et al. (Held et al., 1999; Held et al., 1998) developed a recombinant E. coli strain expressing hydroxybiphenyl 3-monooxygenase for the conversion of 2-substituted phenols into 3-substituted catechols, which are difficult to synthesize chemically. During production of 3-phenylcatechol, in situ product removal prevented toxic effects on the whole-cell biocatalyst. Isolated hydroxybiphenyl 3-monooxygenase has been used in organic-aqueous reaction media in combination with enzymatic coenzyme regeneration (Lutz et al., 2002). Directed evolution has been employed to improve and broaden the substrate specificity and efficiency of this monooxygenase (A. Meyer et al., 2002b; A. Meyer et al., 2002). 6.3 Monooxygenases involved in desulfurization Combustion of sulfur-containing fuels such as diesel leads to formation of sulfur oxides, which are air pollutants. Until 70 % of the sulfur content of diesel oil is presented as dibenzothiophenes (DBTs). The conventional chemical hydrodesulphurization process is not adequate for complete removal of these compounds (Abbad-Andaloussi et al., 2003). Therefore, biocatalytic processes using multi-component flavin monooxygenases may find application in the desulfurization of fossil fuels (Gray et al., 2003). Different approaches have been used to engineer strains with improved desulfurization activity. Overexpression of DszD (see § 4.3) or another flavin reductase in E. coli and P. putida enhanced the overall rate of desulfurization as compared to the use of wild-type strains (Galan et al., 2000; Reichmuth et al., 2000). To circumvent catabolite repression of the dsz gene cluster (see § 1.4.3) by sulfonate, cysteines or methionines, these genes have been placed under control of the tac promotor in various Pseudomonas strains (Gallardo et al., 1997). Furthermore, activities towards (highly) alkylated DBTs have been improved using a chemostat approach (Arensdorf et al., 2002) and gene shuffling (Coco et al., 2001). 6.4 Cyclohexanone monooxygenase From all the flavoprotein monooxygenases, BVMOs have been studied most extensively for their biotechnological applications. BVMOs typically can deal with a large number of different substrates while exhibiting an exquisite enantio- and/or regioselectivity. A nice example of the applicability of a BVMO was recently given by Alphand et al. (Alphand et al., 2003). They demonstrated that E. coli cells expressing cyclohexanone monooxygenase could be used to produce industrially relevant lactones at multi-gram scale. Currently, more than 100 substrates have been described for cyclohexanone monooxygenase (Mihovilovic et al., 2002b).

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7. AIM AND OUTLINE OF THIS THESIS Until the work described in this thesis started in 1998, only BVMOs primarily acting on aliphatic (cyclic) ketones had been explored. However, several microbial strains were isolated in the past that displayed Baeyer-Villiger monooxygenase activity towards aromatic compounds. A number of bacterial species were found to degrade acetophenones via an initial Baeyer-Villiger reaction (Cripps, 1975; Cripps et al., 1978; Darby et al., 1987; Havel and Reineke, 1993; Higson and Focht, 1990). In our laboratory, the interest in BVMOs acting on aromatic compounds was fuelled by results obtained from an IOP project. It involved the synthesis of substituted acylated catechols using microorganisms as a Baeyer-Villiger catalyst that acts on acetophenone derivatives (Figure 4). OH O

R1

OHO

O

R1

BVMO

substituted acetophenone acylated catechol

Figure 4. Production of substituted acylated catechols using a BVMO. These acylated catechols (or protected catechols) are difficult to produce chemically and could be of interest for industry as intermediates for the synthesis of pharmaceuticals (Moonen et al., 2001). The strains Arthrobacter M5 (Havel and Reineke, 1993), Alcaligenes sp. ACA and Pseudomonas fluorescens ACB (Higson and Focht, 1990) were studied for their ability to convert acetophenones into their corresponding esters. From these studies (unpublished work by J. van der Ven) P. fluorescens ACB was found to be the most promising candidate for further investigation. Unfortunately, an esterase that was co-expressed with the monooxygenase, prevented accumulation of the desired products. To overcome this, the gene encoding the BVMO should be cloned into a more suitable host. The cloned gene can also be used to achieve overexpression of the monooxygenase which will facilitate isolation of this BVMO in sufficient amounts for biochemical characterization studies. These objectives are the topic of the PhD-project described in this thesis. The project was supported by the Council for Chemical Sciences of the Netherlands Organization for Scientific Research (CW-NWO), division ‘Procesvernieuwing voor een schoner milieu’. Before the start of the project, both 4-hydroxyacetophenone monooxygenase (HAPMO) and 4-hydroxyphenyl acetate esterase were purified from P. fluorescens ACB and their N-terminal sequences were determined. The first goal was to clone the HAPMO gene to make heterologous expression possible. Besides obtaining a novel BVMO for biotechnological applications, the project aimed at understanding the sequence-function relationship of BVMOs. A biochemical characterization and sequence analysis

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of HAPMO in combination with mutagenesis studies should lead to increased understanding of BVMO functioning. Chapter 2 describes the purification and characterization of HAPMO. The gene was cloned in E. coli and the enzyme could be overexpressed. Primary sequence analysis revealed that HAPMO contains two Rossmann-fold motifs that are involved in binding of the ADP-moieties of FAD and NADPH. Due to an additional N-terminal domain, the HAPMO subunit is significantly larger (72 kDa) than other characterized BVMOs (typically 60 kDa). Chapter 3 describes the discovery of a specific Type I BVMO sequence motif (FXGXXXHXXXW[P/D]) that can be used to identify BVMO sequences in the sequence databases. The roles of the conserved histidine and tryptophan residues were investigated by replacing these residues with an alanine. It was found that mutant H296A was nearly inactive while mutants W300A and W300Y could not be expressed as soluble proteins. This chapter also describes a novel superfamily embracing three families of FAD-dependent monooxygenases: the flavin-containing monooxygenases (FMOs), the N-hydroxylating monooxygenases (NMOs) and the BVMOs. All these single-polypeptide enzymes share FAD and NADPH dependency and have a similar sequence organization. In Chapter 4 the substrate range of HAPMO is explored. It was found that HAPMO is most efficient in converting aromatic ketones, but it can also oxidize aliphatic and heterocyclic ketones. A common substrate for many BVMOs, bicycloheptenone, is also converted by HAPMO. Besides Baeyer-Villiger oxidations, BVMOs also catalyze highly enantioselective sulfoxidations. In Chapter 5 the function of the N-terminal domain of HAPMO is explored. As mentioned above, HAPMO contains an additional N-terminal domain when compared with other BVMOs. Randomly truncated mutants and site-directed mutants were constructed and their properties were studied. Removal of only nine amino-terminal residues drastically decreased the thermostability of HAPMO. The hydrodynamic behavior of the truncated mutants was different from wild-type HAPMO as the mutants monomerize more easily. The results show that this additional N-terminal domain of HAPMO is involved in increasing the thermostability of the enzyme, likely via dimerization. Chapter 6 deals with the coenzyme specificity of HAPMO. The enzyme has a 400-fold preference for NADPH above NADH. Based on an alignment study of Type I BVMOs and related enzymes, mutagenesis experiments were performed to identify amino acid residues that are involved in coenzyme recognition. It is shown that the results can be extrapolated to cyclohexanone monooxygenases. Chapter 7 describes the characteristics of recently cloned Type I BVMOs. Rather than discussing all possible reactions with these enzymes, this review focuses on the substrate ranges of the biocatalysts. Special emphasis is given on finding novel BVMOs from genomes. Furthermore, alternative enzymes are discussed that could catalyze a Baeyer-Villiger reaction. In Chapter 8 general conclusions are given and the results described in this thesis are discussed.

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