Oxidative cyclization of N-methyl-dopa by a fungal flavoenzyme of the amine oxidase family

Majd Lahham, Tea Pavkov-Keller, Michael Fuchs, Johannes Niederhauser, Gabriel Chalhoub, Bastian Daniel, Wolfgang Kroutil, Karl Gruber, Peter Macheroux

Research output: Contribution to journalArticleResearchpeer-review

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-Nmethyl-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.

Original languageEnglish
Pages (from-to)17021-17032
Number of pages12
JournalJournal of Biological Chemistry
Volume293
Issue number44
DOIs
Publication statusPublished - 1 Jan 2018

Fingerprint

Dihydroxyphenylalanine
Cyclization
Amines
Oxidoreductases
Stereoisomerism
Genes
Crystal structure
Biosynthesis
Methyltyrosines
Tyrosine
Substrates
Isoquinolines
Flavoproteins
Oxidation
Flavin-Adenine Dinucleotide
Dronabinol
Aspergillus fumigatus
Aspergillus
Enzymes
Multigene Family

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Oxidative cyclization of N-methyl-dopa by a fungal flavoenzyme of the amine oxidase family. / Lahham, Majd; Pavkov-Keller, Tea; Fuchs, Michael; Niederhauser, Johannes; Chalhoub, Gabriel; Daniel, Bastian; Kroutil, Wolfgang; Gruber, Karl; Macheroux, Peter.

In: Journal of Biological Chemistry, Vol. 293, No. 44, 01.01.2018, p. 17021-17032.

Research output: Contribution to journalArticleResearchpeer-review

Lahham M, Pavkov-Keller T, Fuchs M, Niederhauser J, Chalhoub G, Daniel B et al. Oxidative cyclization of N-methyl-dopa by a fungal flavoenzyme of the amine oxidase family. Journal of Biological Chemistry. 2018 Jan 1;293(44):17021-17032. https://doi.org/10.1074/jbc.RA118.004227
Lahham, Majd ; Pavkov-Keller, Tea ; Fuchs, Michael ; Niederhauser, Johannes ; Chalhoub, Gabriel ; Daniel, Bastian ; Kroutil, Wolfgang ; Gruber, Karl ; Macheroux, Peter. / Oxidative cyclization of N-methyl-dopa by a fungal flavoenzyme of the amine oxidase family. In: Journal of Biological Chemistry. 2018 ; Vol. 293, No. 44. pp. 17021-17032.
@article{58820ace8ab24ba38fd3f633f21eb9d5,
title = "Oxidative cyclization of N-methyl-dopa by a fungal flavoenzyme of the amine oxidase family",
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 {\AA} 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-Nmethyl-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.",
author = "Majd Lahham and Tea Pavkov-Keller and Michael Fuchs and Johannes Niederhauser and Gabriel Chalhoub and Bastian Daniel and Wolfgang Kroutil and Karl Gruber and Peter Macheroux",
year = "2018",
month = "1",
day = "1",
doi = "10.1074/jbc.RA118.004227",
language = "English",
volume = "293",
pages = "17021--17032",
journal = "The Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "44",

}

TY - JOUR

T1 - Oxidative cyclization of N-methyl-dopa by a fungal flavoenzyme of the amine oxidase family

AU - Lahham, Majd

AU - Pavkov-Keller, Tea

AU - Fuchs, Michael

AU - Niederhauser, Johannes

AU - Chalhoub, Gabriel

AU - Daniel, Bastian

AU - Kroutil, Wolfgang

AU - Gruber, Karl

AU - Macheroux, Peter

PY - 2018/1/1

Y1 - 2018/1/1

N2 - 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-Nmethyl-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.

AB - 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-Nmethyl-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.

UR - http://www.scopus.com/inward/record.url?scp=85056061877&partnerID=8YFLogxK

U2 - 10.1074/jbc.RA118.004227

DO - 10.1074/jbc.RA118.004227

M3 - Article

VL - 293

SP - 17021

EP - 17032

JO - The Journal of Biological Chemistry

JF - The Journal of Biological Chemistry

SN - 0021-9258

IS - 44

ER -