Abstract
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.
Highlights
Lines of diverse structure, such as protopine, sanguinarine, and berberine, in a series of enzyme-catalyzed transformations [2]
(BBE), heterologous expression in a bacterial host3 as well as in S. cerevisiae were not met with much success leading either to insoluble protein or to very small amounts of soluble and active enzyme [7]
Higher amounts of active berberine bridge enzyme (BBE) could be generated in insect cell culture using the baculovirus expression system [8], the amounts obtained were still not sufficient for a detailed biochemical, mechanistic, and structural investigation of the enzyme
Summary
SCHEME 1 lighted by the fact that the gene possesses an N-terminal signal peptide as well as a vacuolar sorting determinant [7, 9]. Active enzyme requires covalently bound FAD and is N-glycosylated. We report the construction of a new expression system for BBE in the methylotrophic yeast Pichia pastoris by using the secretory pathway of this organism. This approach yields large amounts of highly active BBE enabling us to study some basic properties of the enzyme. In the course of this characterization, we could demonstrate that the enzyme contains an FAD cofactor, which is covalently linked to a histidine (His-104) and a cysteine (Cys-166) residue, as recently reported for glucooligosaccharide oxidase from Acremonium strictum [10, 11]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
