N-methyldopa

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.

Production, purification, and characterization of FsqB

FsqB was expressed in Escherichia coli yielding 1 mg of FsqB from 1 g of pellet after purification by nickel–nitrilotriacetic acid affinity chromatography. For crystallization, the protein was further purified by means of size-exclusion 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 (A). The bathochromic shift of the long wavelength absorption maximum by ∼10 nm compared with free FAD is most likely due to the monocovalent attachment of the 8α-methyl group to cysteine residue 414. Similar UV-visible absorption spectra were previously reported for human dimethylglycine dehydrogenase (hDMGDH) () and N-methyl-tryptophan oxidase (), which feature the same covalent linkage. The extinction coefficient of the FAD bound to FsqB was calculated to 12,350 m−1 cm−1. Consumption of N-methyl-dopa was investigated from pH 4 to 9 using an oxygen sensor to monitor the reaction yielding a bell-shaped profile with an optimum at pH 7.6. Photoreduction of FsqB led to the formation of a stable flavin semiquinone with an absorption maximum at 396 nm. Further reduction of the flavin semiquinone to the hydroquinone could not be achieved by light. The flavin semiquinone was reoxidized very slowly by molecular oxygen reaching completion after ∼70 min (B).

Kinetic parameters of FsqB

Following product analysis, we also determined pre–steady-state parameters in the stopped-flow apparatus for N-methyl-dopa, rac-N-methyl-meta-tyrosine, and l-N-methyl-tyrosine. The highest rate of flavin reduction was observed with (racemic) N-methyl-dopa, whereas (racemic) N-methyl-meta-tyrosine and l-N-methyl-tyrosine were 10–25 times slower (the kred values given in were measured at a single fixed concentration of 2.5 mm). A more detailed analysis of the reduction of FsqB using rac-N-methyl-dopa or the l-stereoisomer yielded limiting rates of 51 ± 3 and 21 ± 3 s−1, as well as dissociation constants of 9.2 ± 0.7 and 8.0 ± 1.3 mm, respectively (A and ). Thus, the limiting rate for l-N-methyl-dopa is only ∼2.5-fold lower than for the racemate, indicating that both stereoisomers are accepted by the enzyme. Furthermore, this result also suggests that the enzyme has a preference for d-N-methyl-dopa (commercially not available). To distinguish the rate of reduction for the d- and l-stereoisomer, we repeated the pre–steady-state measurements at a lower concentration of rac-N-methyl-dopa (i.e. 0.2 mm) and obtained a clear biphasic behavior with observed rates of 1.63 ± 0.04 and 0.61 ± 0.01 s−1, respectively, presumably reflecting the rate of reduction for the two stereoisomers in the racemic mixture. This 2.5-fold difference is similar to the difference observed for the racemate compared with the l-stereoisomer (), supporting our conclusion that the d-stereoisomer is a slightly better substrate than the l-stereoisomer. Notably, at substrate concentrations above 0.2 mm, the reaction showed essentially monophasic behavior because the fast-reacting enantiomer outcompetes the slower one. Interestingly, reduction of the FAD cofactor was not observed with d-N-methyl-tyrosine, suggesting that FsqB exhibits strict stereo-preference for the l-N-methyl-tyrosine stereoisomer (S configuration) in contrast to what was found with N-methyl-dopa.

Kinetic parameters of FsqB

Steady-state kinetics were determined for N-methyl-dopa (racemate and the l-stereoisomer), l-N-methyl-tyrosine, and rac-N-methyl-meta-tyrosine using oxygen depletion to monitor enzyme turnover. As shown in B, enzyme activity was a function of substrate concentration reaching saturation at ≈8 s−1. Similarly, the Km for the racemate and the l-enantiomer of N-methyl-dopa differed only slightly, i.e. 0.16 ± 0.01 and 0.27 ± 0.02 mm, respectively. For l-N-methyl-tyrosine and rac-N-methyl-meta-tyrosine, saturation was reached at ≈4 and ≈3.5 s−1, respectively. The Km for these substrates was 2.1 ± 0.2 and 1.6 ± 0.6 mm, respectively. summarizes the kinetic parameters for all substrates oxidized by FsqB. As judged by the resulting kcat/Km values, N-methyl-dopa (racemate and the l-enantiomere) is more efficiently oxidized by FsqB than l-N-methyl-tyrosine and rac-N-methyl-meta-tyrosine ().