During the last decades a large number of flavin-dependent monooxygenases have been isolated and studied. This has revealed that flavoprotein monooxygenases are able to catalyze a remarkable wide variety of oxidative reactions such as regioselective hydroxylations and enantioselective sulfoxidations. These oxidation reactions are often difficult, if not impossible, to be achieved using chemical approaches. Analysis of the available genome sequences has indicated that many more flavoprotein monooxygenases exist and await biocatalytic exploration. Based on the known biochemical properties of a number of flavoprotein monooxygenases and sequence and structural analyses, flavoprotein monooxygenases can be classified into six distinct flavoprotein monooxygenase subclasses. This review provides an inventory of known flavoprotein monooxygenases belonging to these different enzyme subclasses. Furthermore, the biocatalytic potential of a selected number of flavoprotein monooxygenases is highlighted.

Baeyer^Villiger monooxygenases (BVMOs) form a distinct class of flavoproteins that catalyze the insertion of an oxygen atom in a C^C bond using dioxygen and NAD(P)H. Using newly characterized BVMO sequences, we have uncovered a BVMO-identifying sequence motif: FXGXXXHXXXW(P/D). Studies with site-directed mutants of 4-hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB suggest that this fingerprint sequence is critically involved in catalysis. Further sequence analysis showed that the BVMOs belong to a novel superfamily that comprises three known classes of FAD-dependent monooxygenases: the so-called flavin-containing monooxygenases (FMOs), the N-hydroxylating monooxygenases (NMOs), and the BVMOs. Interestingly, FMOs contain an almost identical sequence motif when compared to the BVMO sequences: FXGXXXHXXX(Y/F). Using these novel amino acid sequence fingerprints, BVMOs and FMOs can be readily identified in the protein sequence databank. ß

EtaA is a newly identified FAD-containing monooxygenase that is responsible for activation of several thioamide prodrugs in Mycobacterium tuberculosis. It was found that purified EtaA displays a remarkably low activity with the antitubercular prodrug ethionamide. Hinted by the presence of a Baeyer-Villiger monooxygenase sequence motif in the EtaA sequence, we have been able to identify a large number of novel EtaA substrates. It was discovered that the enzyme converts a wide range of ketones to the corresponding esters or lactones via a Baeyer-Villiger reaction, indicating that EtaA represents a Baeyer-Villiger monooxygenase. With the exception of aromatic ketones (phenylacetone and benzylacetone), long-chain ketones (e.g. 2-hexanone and 2-dodecanone) also are converted. EtaA is also able to catalyze enantioselective sulfoxidation of methyl-ptolylsulfide. Conversion of all of the identified substrates is relatively slow with typical k cat values of around 0.02 s ؊1 . The best substrate identified so far is phenylacetone (K m ‫؍‬ 61 M, k cat ‫؍‬ 0.017 s ؊1 ). Redox monitoring of the flavin cofactor during turnover of phenylacetone indicates that a step in the reductive half-reaction is limiting the rate of catalysis. Intriguingly, EtaA activity could be increased by one order of magnitude by adding bovine serum albumin. This reactivity and substrate acceptance-profiling study provides valuable information concerning this newly identified prodrug activator from M. tuberculosis.

The syndrome of leukocyte adhesion deficiency (LAD) combined with a severe Glanzmann-type bleeding disorder has been recognized as a separate disease entity. The variability in clinical and cell biological terms has remained largely unclear. We present data on 9 cases from 7 unrelated families, with 3 patients being actively followed for more than 12 years. The disease entity, designated LAD-1/ variant syndrome, presents early in life and consists of nonpussing infections from bacterial and fungal origin, as well as a severe bleeding tendency. This is compatible with 2 major blood cell types contributing to the clinical symptoms (ie, granulocytes and platelets). In granulocytes of the patients, we found adhesion and chemotaxis defects, as well as a defect in NADPH oxidase activity triggered by unopsonized zymosan. This last test can be used as a screening test for the syndrome. Many proteins and genes involved in adhesion and signaling, including small GTPases such as Rap1 and Rap2 as well as the major Rap activityregulating molecules, were normally present. Moreover, Rap1 activation was intact in patients' blood cells. Defining the primary defect awaits genetic linkage analysis, which may be greatly helped by a more precise understanding and awareness of the disease combined with the early identification of affected patients.

A novel flavoprotein that catalyses the NADPH-dependent oxidation of 4-hydroxyacetophenone to 4-hydroxyphenyl acetate, was purified to homogeneity from Pseudomonas fluorescens ACB. Characterization of the purified enzyme showed that 4-hydroxyacetophenone monooxygenase (HAPMO) is a homodimer of < 140 kDa with each subunit containing a noncovalently bound FAD molecule. HAPMO displays a tight coupling between NADPH oxidation and substrate oxygenation. Besides 4-hydroxyacetophenone a wide range of other acetophenones are readily converted via a Baeyer±Villiger rearrangement reaction into the corresponding phenyl acetates. The P. fluorescens HAPMO gene (hapE) was characterized. It encoded a 640 aminoacid protein with a deduced mass of 71 884 Da. Except for an N-terminal extension of < 135 residues, the sequence of HAPMO shares significant similarity with two known types of Baeyer±Villiger monooxygenases: cyclohexanone monooxygenase (27±33% sequence identity) and steroid monooxygenase (33% sequence identity). The HAPMO sequence contains several sequence motifs indicative for the presence of two Rossman fold domains involved in FAD and NADPH binding. The functional role of a recently identified flavoprotein sequence motif (ATG) was explored by site-directed mutagenesis. Replacement of the strictly conserved glycine (G490) resulted in a dramatic effect on catalysis. From a kinetic analysis of the G490A mutant it is concluded that the observed sequence motif serves a structural function which is of importance for NADPH binding.

The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH-and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this flavin-containing monooxygenase. On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO. In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides. Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO. To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied. The monooxygenase preferably converted (1R,5S)-bicyclohept-2-en-6-one, with an enantiomeric ratio (E) of 20, thus enabling kinetic resolution to obtain the (1S,5R) enantiomer. Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (ee) for the two lactones obtained [77% ee for (1S,5R)-2 and 34% ee for (1R,5S)-3]. Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (S)-sulfoxides (ee > 99%). The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications.In nature, many oxygenation reactions are carried out by flavin-dependent monooxygenases (24). The diversity of conversions that can be catalyzed is large; the reactions include Baeyer-Villiger reactions, aromatic hydroxylations, sulfoxidations, amine oxidations, and epoxidations. Many of these conversions occur with high enantio-and/or regioselectivity. The variety of reactivity is also reflected in the diverse physiological processes in which these enzymes play a prominent role, including xenobiotic compound metabolism in humans (49), biosynthesis of toxins (21), and pollutant degradation by bacteria (46).Enantio-and regioselective oxygenations are often difficult to achieve by chemical means, while these types of reactions can lead to valuable optically active compounds. Due to their exquisite regio-and/or enantioselectivity and catalytic efficiency, flavin-dependent monooxygenases are useful biocatalysts for the synthesis of a variety of fine chemicals (12,35,41,47). However, so far, only a limited number of flavin-dependent monooxygenase genes have been cloned and overexpressed, which has limited the application of these biocatalysts for synthetic processes.The Baeyer-Villiger reaction, i.e., the oxidation of ketones or aldehydes by peroxides resulting in oxygen insertion adjacent to the carbonyl group (2), has many applications in organic ...

The Baeyer-Villiger monooxygenase (BVMO), 4-hydroxyacetophenone monooxygenase (HAPMO), uses NADPH and O 2 to oxidize a variety of aromatic ketones and sulfides. The FAD-containing enzyme has a 700-fold preference for NADPH over NADH. Sequence alignment with other BVMOs, which are all known to be selective for NADPH, revealed three conserved basic residues, which could account for the observed coenzyme specificity. The corresponding residues in HAPMO (Arg339, Lys439 and Arg440) were mutated and the properties of the purified mutant enzymes were studied. For Arg440 no involvement in coenzyme recognition could be shown as mutant R440A was totally inactive. Although this mutant could still be fully reduced by NADPH, no oxygenation occurred, indicating that this residue is crucial for completing the catalytic cycle of HAPMO. Characterization of several Arg339 and Lys439 mutants revealed that these residues are indeed both involved in coenzyme recognition. Mutant R339A showed a largely decreased affinity for NADPH, as judged from kinetic analysis and binding experiments. Replacing Arg339 also resulted in a decreased catalytic efficiency with NADH.Mutant K439A displayed a 100-fold decrease in catalytic efficiency with NADPH, mainly caused by an increased K m . However, the efficiency with NADH increased fourfold. Saturation mutagenesis at position 439 showed that the presence of an asparagine or a phenylalanine improves the catalytic efficiency with NADH by a factor of 6 to 7. All Lys439 mutants displayed a lower affinity for AADP + , confirming a role of the lysine in recognizing the 2¢-phosphate of NADPH. The results obtained could be extrapolated to the sequence-related cyclohexanone monooxygenase. Replacing Lys326 in this BVMO, which is analogous to Lys439 in HAPMO, again changed the coenzyme specificity towards NADH. These results indicate that the strict NADPH dependency of this class of monooxygenases is based upon recognition of the coenzyme by several basic residues.

The catabolism of 4-hydroxyacetophenone in Pseudomonas fluorescens ACB is known to proceed through the intermediate formation of hydroquinone. Here, we provide evidence that hydroquinone is further degraded through 4-hydroxymuconic semialdehyde and maleylacetate to ␤-ketoadipate. The P. fluorescens ACB genes involved in 4-hydroxyacetophenone utilization were cloned and characterized. Sequence analysis of a 15-kb DNA fragment showed the presence of 14 open reading frames containing a gene cluster (hapCDEFGHIBA) of which at least four encoded enzymes are involved in 4-hydroxyacetophenone degradation: 4-hydroxyacetophenone monooxygenase (hapA), 4-hydroxyphenyl acetate hydrolase (hapB), 4-hydroxymuconic semialdehyde dehydrogenase (hapE), and maleylacetate reductase (hapF). In between hapF and hapB, three genes encoding a putative intradiol dioxygenase (hapG), a protein of the Yci1 family (hapH), and a [2Fe-2S] ferredoxin (hapI) were found. Downstream of the hap genes, five open reading frames are situated encoding three putative regulatory proteins (orf10, orf12, and orf13) and two proteins possibly involved in a membrane efflux pump (orf11 and orf14). Upstream of hapE, two genes (hapC and hapD) were present that showed weak similarity with several iron(II)-dependent extradiol dioxygenases. Based on these findings and additional biochemical evidence, it is proposed that the hapC and hapD gene products are involved in the ring cleavage of hydroquinone.