Biochemical evidence that berberine bridge enzyme belongs to a novel family of flavoproteins containing a bi-covalently attached FAD cofactor.
Berberine bridge enzyme (BBE) is involved in the transformation of (S)-reticuline to (S)-scoulerine in benzophenanthridine alkaloid biosynthesis of plants. In this report, we describe the high level expression of BBE encoded by the gene from Eschscholzia californica (California poppy) in the methylotrophic yeast Pichia pastoris employing the secretory pathway of the host organism. Using a two-step chromatographic purification protocol, 120 mg of BBE could be obtained from 1 liter of fermentation culture. The purified protein exhibits a turnover number for substrate conversion of 8.2 s(-1). The recombinant enzyme is glycosylated and carries a covalently attached FAD cofactor. In addition to the previously known covalent attachment of the 8alpha-position of the flavin ring system to a histidine (His-104), we could also demonstrate that a covalent linkage between the 6-position and a thiol group of a cysteine residue (Cys-166) is present in BBE. The major evidence for the occurrence of a bi-covalently attached FAD cofactor is provided by N-terminal amino acid sequencing and mass spectrometric analysis of the isolated flavin-containing peptide. Furthermore, it could be shown that anaerobic photoirradiation leads to cleavage of the linkage between the 6-cysteinyl group yielding 6-mercaptoflavin and a peptide with the cysteine residue replaced by alanine due to breakage of the C-S bond. Overall, BBE is shown to exhibit typical flavoprotein oxidase properties as exemplified by the occurrence of an anionic flavin semiquinone species and formation of a flavin N(5)-sulfite adduct.
Winkler
Andreas
A
Graz University of Technology, Institute of Biochemistry, Petersgasse 12/II, A-8010 Graz, Austria.
Hartner
Franz
F
Research Centre Applied Biocatalysis, c/o Graz University of Technology, Institute of Molecular Biotechnology, A-8010 Graz, Austria.
Kutchan
Toni M
TM
Abteilung Naturstoff-Biotechnologie, Leibniz-Institut für Pflanzenbiochemie, D-06120 Halle/Saale, Germany.
Glieder
Anton
A
Research Centre Applied Biocatalysis, c/o Graz University of Technology, Institute of Molecular Biotechnology, A-8010 Graz, Austria.
Macheroux
Peter
P
Graz University of Technology, Institute of Biochemistry, Petersgasse 12/II, A-8010 Graz, Austria. Electronic address: peter.macheroux@tugraz.at.
D000595:Amino Acid Sequence; D000838:Anions / Q000737:chemistry; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005420:Flavoproteins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry* / Q000502:physiology*; D010207:Papaver / Q000201:enzymology* / Q000235:genetics; D010455:Peptides / Q000737:chemistry; D010843:Pichia; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000838:Anions; D005420:Flavoproteins; D010455:Peptides; D005182:Flavin-Adenine Dinucleotide; D010088:Oxidoreductases; C071690:reticuline oxidase; D010089:Oxidoreductases, N-Demethylating