Baeyer-Villiger monooxygenases represent useful biocatalytic tools, as they can catalyze reactions which are difficult to achieve using chemical means. However, only a limited number of these atypical monooxygenases are available in recombinant form. Using a recently described protein sequence motif, a putative Baeyer-Villiger monooxygenase (BVMO) was identified in the genome of the thermophilic actinomycete Thermobifida fusca. Heterologous expression of the respective protein in Escherichia coli and subsequent enzyme characterization showed that it indeed represents a BVMO. The NADPH-dependent and FAD-containing monooxygenase is active with a wide range of aromatic ketones, while aliphatic substrates are also converted. The best substrate discovered so far is phenylacetone (k(cat) = 1.9 s(-1), K(M) = 59 microM). The enzyme exhibits moderate enantioselectivity with alpha-methylphenylacetone (enantiomeric ratio of 7). In addition to Baeyer-Villiger reactions, the enzyme is able to perform sulfur oxidations. Different from all known BVMOs, this newly identified biocatalyst is relatively thermostable, displaying an activity half-life of 1 day at 52 degrees C. This study demonstrates that, using effective annotation tools, genomes can efficiently be exploited as a source of novel BVMOs.

A gene encoding an alditol oxidase was found in the genome of Streptomyces coelicolor A3(2). This newly identified oxidase, AldO, was expressed at extremely high levels in Escherichia coli when fused to maltose-binding protein. AldO Carbohydrate oxidases are highly valuable biocatalysts for analytical and synthetic purposes. Chemical methods cannot compete with the exquisite regio-and/or enantioselectivity by which these enzymes oxidize polyols. Applications in which carbohydrate oxidases are used are, for example, biosensors for blood sugar, synthetic routes toward chiral building blocks, sweeteners, and flavors. Oxidase-mediated catalysis also leads to formation of hydrogen peroxide, a property that is used in a number of applications, such as bleaching processes and wastewater treatment (1, 2). At present, only a limited number of carbohydrate oxidases have been identified, which restricts the biocatalytic exploitation of this class of redox enzymes. The best known representative is glucose oxidase, which in fact is the most widely applied redox enzyme.Besides galactose oxidase, which contains copper as cofactor, all presently known oxidases acting on carbohydrates contain a flavin cofactor. Examples of such flavoprotein oxidases are glucose oxidase, L-gulono-␥-lactone oxidase, xylitol oxidase, hexose oxidase, lactose oxidase, glucooligosaccharide oxidase, and pyranose oxidase. Except for glucose oxidase and pyranose oxidase, all of these oxidases belong to a specific group of flavoproteins, the vanillyl-alcohol oxidase (VAO) 2 family. Members of this family share a similar overall structure consisting of two domains (3). One domain binds the adenine part of the FAD cofactor and is called the FAD-binding domain, whereas the other, called the cap domain, covers the isoalloxazine moiety of the cofactor and forms the major part of the active site around the isoalloxazine ring system. A special feature of this flavoprotein family is the fact that a relatively large number of VAO members bind the FAD cofactor in a covalent manner. This is also the case for all of the above mentioned VAO-type carbohydrate oxidases. In fact, the recent elucidation of the structure of glucooligosaccharide oxidase has revealed the first example where a flavin cofactor is covalently linked to two amino acid residues (4). It has been shown that a covalent FAD-protein linkage can have a significant effect on the redox behavior of the flavin cofactor (e.g. increasing the redox potential) (5). This is in line with the observation that most VAO-type covalent flavoproteins act as an oxidase (6). In these cases, the FAD cofactor is typically tethered to a histidine residue, and this linking histidine can be readily identified by sequence motif recognition. Hence, the ability to identify covalent VAO homologs by sequence analysis can be used as a tool to find novel oxidase genes.Most of the characterized carbohydrate oxidases have been isolated from fungi, whereas only two from bacterial origin have been described (7,8). Here we describe the...

Oxygenases form an interesting class of biocatalysts, as they typically perform oxygenations with exquisite chemo-, regio-, and/or enantioselectivity. It has been observed that, once heterologously expressed in Escherichia coli, some oxygenases are able to form the blue pigment indigo. We have exploited this characteristic to screen a metagenomic library derived from loam soil and identified a novel oxygenase. This oxygenase shows 50% sequence identity with styrene monooxygenases from pseudomonads (StyA). Only a limited number of homologs can be found in the genome sequence database, indicating that this biocatalyst is a member of a relatively small family of bacterial monooxygenases. The newly identified monooxygenase catalyzes the epoxidation of styrene and styrene derivatives and forms the corresponding (S)-epoxides with excellent enantiomeric excess [e.g., (S)-styrene oxide is formed with >99% enantiomeric excess, ee] and therefore is named styrene monooxgenase subunit A (SmoA). SmoA shows high enantioselectivity towards aromatic sulfides [e.g., (R)-ethyl phenyl sulfoxide is formed with 92% ee]. This excellent enantioselectivity in combination with the moderate sequence identity forms a clear indication that SmoA from a metagenomic origin represents a new enzyme within the small family of styrene monooxygenases.Oxygenases are of growing interest for biotechnological applications (5, 38). These oxidative biocatalysts are able to insert one or two oxygen atoms into a substrate molecule. In order to do so, they usually need NAD(P)H as an electron donating coenzyme, while a metal or organic cofactor is required for oxygen functionalization. A feature that makes these biocatalysts of special interest is their ability to perform oxygenations in a chemo-, regio-, and/or enantioselective manner (13, 38). Often, selectivities are observed that cannot be rivaled by chemical approaches. Due to their selectivity and ability to use oxygen as a cheap and environmentally friendly oxidant, oxygenases form an important class of enzymes which can be applied for the biocatalytic oxidation of several compounds. The need for expensive coenzymes is no longer considered a hurdle for such industrial applications, since biocatalytic systems employing whole cells can be used which circumvent expensive coenzyme regeneration procedures (36).Oxygenases are found in all kingdoms of life but especially in bacteria. Their physiological role is often related to degradation of toxic compounds or the synthesis of secondary metabolites (40). The classical approach to finding novel oxygenases with biocatalytic potential is therefore to screen for organisms which are able to grow on toxic compounds, like aromatics, or to use cultures which are enriched with these kinds of compounds. However, this method only focuses on the culturable portion of organisms which contain putative oxygenases, and it is estimated that more than 99% of all microbes are not cultivable (1) and therefore not accessible as a source for finding novel biocatalysts. With the...

A gene encoding a putrescine oxidase (PuO Rh , EC 1.4.3.10) was identified from the genome of Rhodococcus erythropolis NCIMB 11540. The gene was cloned in the pBAD vector and overexpressed at high levels in Escherichia coli. The purified enzyme was shown to be a soluble dimeric flavoprotein consisting of subunits of 50 kDa and contains non-covalently bound flavin adenine dinucleotide as a cofactor. From all substrates, the highest catalytic efficiency was found with putrescine (K M =8.2 μM, k cat = 26 s −1 ). PuO Rh accepts longer polyamines, while short diamines and monoamines strongly inhibit activity. PuO Rh is a reasonably thermostable enzyme with t 1/2 at 50°C of 2 h. Based on the crystal structure of human monoamine oxidase B, we constructed a model structure of PuO Rh , which hinted to a crucial role of Glu324 for substrate binding. Mutation of this residue resulted in a drastic drop (five orders of magnitude) in catalytic efficiency. Interestingly, the mutant enzyme showed activity with monoamines, which are not accepted by wt-PuO Rh .

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