Biological oxidations form the basis of life on earth by utilizing organic compounds as electron donors to drive the generation of metabolic energy carriers, such as ATP. Oxidative reactions are also important for the biosynthesis of complex compounds, i.e. natural products such as alkaloids that provide vital benefits for organisms in all kingdoms of life. The vitamin B-derived cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) enable an astonishingly diverse array of oxidative reactions that is based on the versatility of the redox-active isoalloxazine ring. The family of FAD-linked oxidases can be divided into subgroups depending on specific sequence features in an otherwise very similar structural context. The sub-family of berberine bridge enzyme (BBE)-like enzymes has recently attracted a lot of attention due to the challenging chemistry catalyzed by its members and the unique and unusual bi-covalent attachment of the FAD cofactor. This family is the focus of the present review highlighting recent advancements into the structural and functional aspects of members from bacteria, fungi and plants. In view of the unprecedented reaction catalyzed by the family's namesake, BBE from the California poppy, recent studies have provided further insights into nature's treasure chest of oxidative reactions.

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Flavin-dependent enzymes catalyze many oxidations, including formation of ring structures in natural products. The gene cluster for biosynthesis of fumisoquins, secondary metabolites structurally related to isoquinolines, in the filamentous fungus Aspergillus fumigatus harbors a gene that encodes a flavoprotein of the amine oxidase family, termed fsqB (fumisoquin biosynthesis gene B). This enzyme catalyzes an oxidative ring closure reaction that leads to the formation of isoquinoline products. This reaction is reminiscent of the oxidative cyclization reported for berberine bridge enzyme and tetrahydrocannabinol synthase. Despite these similarities, amine oxidases and berberine bridge enzyme–like enzymes possess distinct structural properties, prompting us to investigate the structure–function relationships of FsqB. Here, we report the recombinant production and purification of FsqB, elucidation of its crystal structure, and kinetic analysis employing five putative substrates. The crystal structure at 2.6 Å resolution revealed that FsqB is a member of the amine oxidase family with a covalently bound FAD cofactor. N-methyl-dopa was the best substrate for FsqB and was completely converted to the cyclic isoquinoline product. The absence of the meta-hydroxyl group, as e.g. in l-N-methyl-tyrosine, resulted in a 25-fold lower rate of reduction and the formation of the demethylated product l-tyrosine, instead of a cyclic product. Surprisingly, FsqB did not accept the d-stereoisomer of N-methyltyrosine, in contrast to N-methyl-dopa, for which both stereoisomers were oxidized with similar rates. On the basis of the crystal structure and docking calculations, we postulate a substrate-dependent population of distinct binding modes that rationalizes stereospecific oxidation in the FsqB active site.

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