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...

Many enzymes use one or more cofactors, such as biotin, heme, or flavin. These cofactors may be bound to the enzyme in a noncovalent or covalent manner. Although most flavoproteins contain a noncovalently bound flavin cofactor (FMN or FAD), a large number have these cofactors covalently linked to the polypeptide chain. Most covalent flavin–protein linkages involve a single cofactor attachment via a histidyl, tyrosyl, cysteinyl or threonyl linkage. However, some flavoproteins contain a flavin that is tethered to two amino acids. In the last decade, many studies have focused on elucidating the mechanism(s) of covalent flavin incorporation (flavinylation) and the possible role(s) of covalent protein–flavin bonds. These endeavors have revealed that covalent flavinylation is a post‐translational and self‐catalytic process. This review presents an overview of the known types of covalent flavin bonds and the proposed mechanisms and roles of covalent flavinylation.

The VAO flavoprotein family is a rapidly growing family of oxidoreductases that favor the covalent binding of the FAD cofactor. In this review we report on the catalytic properties of some newly discovered VAO family members and their mode of flavin binding. Covalent binding of the flavin is a self-catalytic post-translational modification primarily taking place in oxidases. Covalent flavinylation increases the redox potential of the cofactor and thus its oxidation power. Recent findings have revealed that some members of the VAO family anchor the flavin via a dual covalent linkage (6-S-cysteinyl-8alpha-N1-histidyl FAD). Some VAO-type aldonolactone oxidoreductases favor the non-covalent binding of the flavin cofactor. These enzymes act as dehydrogenases, using cytochrome c as electron acceptor.

Alditol oxidase (AldO) from Streptomyces coelicolor A3(2) is a soluble monomeric flavindependent oxidase that performs selective oxidation of the terminal primary hydroxyl group of several alditols. Here, we report the crystal structure of the recombinant enzyme in its native state and in complex with both six-carbon (mannitol and sorbitol) and five-carbon substrates (xylitol). AldO shares the same folding topology of the members of the vanillyl-alcohol oxidase family of flavoenzymes and exhibits a covalently linked FAD which is located at the bottom of a funnel-shaped pocket that forms the active site. The high resolution of the three-dimensional structures highlights a well-defined hydrogen-bonding network that tightly constrains the substrate in the productive conformation for catalysis. Substrate binding occurs through a lock-and-key mechanism and does not induce conformational changes with respect to the ligand-free protein. A network of charged residues is proposed to favor catalysis through stabilization of the deprotonated form of the substrate. A His side chain acts as back door that "pushes" the substratereactive carbon atom toward the N5-C4a locus of the flavin. Analysis of the three-dimensional structure reveals possible pathways for diffusion of molecular oxygen and a small cavity on the re side of the flavin that may host oxygen during FAD reoxidation. These features combined with the tight shape of the catalytic site provide insights into the mechanism of AldO-mediated regioselective oxidation reactions and its substrate specificity. † The financial support by the Italian Ministry of Science (PRIN06 and FIRB programs), and the Petroleum Research Fund (46271-C4), administered by the American Chemical Society, is gratefully acknowledged.‡ Coordinates and structure factors have been deposited with the Protein Data Bank with the accession codes 2vfr, 2vfs, 2vft, 2vfu, and 2vfv.