Abstract
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.
Highlights
The resulting K448A variant was completely devoid of FAD, confirming earlier findings for MSOX that this residue plays an essential role for establishing the covalent bond between the 8␣-methyl group and the corresponding cysteine
Recent fragment molecular orbital and mixed quantum mechanics/molecular mechanics calculations have concluded that a hydride transfer mechanism is the most likely scenario [12, 13]
We assumed that the oxidative cyclization catalyzed by FsqB might be initiated by a hydride transfer, followed by the nucleophilic attack of the catechol moiety of the substrate to yield the isoquinoline product because the electron-rich catechol moiety can be deprotonated at the 3-OH
Summary
FsqB was expressed in Escherichia coli yielding 1 mg of FsqB from 1 g of pellet after purification by nickel–nitrilotriacetic acid affinity chromatography. The UV-visible absorption spectrum of FsqB possesses the typical features of an FAD-containing protein with absorption maxima at 460 and 370 nm (Fig. 1A). The bathochromic shift of the long wavelength absorption maximum by ϳ10 nm compared with free FAD is most likely due to the monocovalent attachment of the 8␣-methyl group to cysteine residue 414. Similar UV-visible absorption spectra were previously reported for human dimethylglycine dehydrogenase (hDMGDH) [5] and N-methyl-tryptophan oxidase [6], which feature the same covalent linkage. Photoreduction of FsqB led to the formation of a stable flavin semiquinone with an absorption maximum at 396 nm. The flavin semiquinone was reoxidized very slowly by molecular oxygen reaching completion after ϳ70 min (Fig. 1B)
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