6-S-Cysteinylation of Bi-covalently Attached FAD in Berberine Bridge Enzyme Tunes the Redox Potential for Optimal Activity

Abstract

A mutagenic analysis of the amino acid residues His-104 and Cys-166, which are involved in the bi-covalent attachment of FAD to berberine bridge enzyme, was performed. Here we present a detailed biochemical characterization of the cysteine link to FAD observed in this recently discovered group of flavoproteins. The C166A mutant protein still has residual activity, but reduced to ϳ6% of the turnover rate observed for wild-type berberine bridge enzyme. A more detailed analysis of single reaction steps by stopped-flow spectrophotometry showed that the reductive half-reaction is greatly influenced by the lack of the 6-S-cysteinyl linkage, resulting in a 370-fold decrease in the rate of flavin reduction. Determination of the redox potentials for both wild type and the C166A mutein revealed that the difference in the redox potential observed can fully account for the change in the kinetic properties. The wild-type protein exhibits a midpoint potential of ؉132 mV, which is the highest redox potential determined for any flavoenzyme so far. Removal of the cysteine linkage to FAD in the C166A mutein leads to a redox potential of ؉53 mV, which is in the expected range for flavoproteins with a single covalent attachment of FAD to a His residue via its 8-␣ position. We also show that the biochemical properties of the mutein resemble that of typical flavoprotein oxidases and that deviations from this behavior observed for the wild type are due to the FAD-6-S-cysteinyl bond. In addition, rapid reaction stopped-flow experiments give no indication for a radical mechanism supporting the direct transfer of a hydride from the substrate to the cofactor. Berberine bridge enzyme (BBE)2 (EC 1.21.3.3) is a central enzyme of alkaloid biosynthesis in plant species capable of producing protopine, protoberberine, and benzophenanthridine alkaloids. Because of the challenging chemical reaction it catalyzes and its involvement in the production of pharmaceutically important chemicals, it has long been of interest to get a detailed understanding of the processes occurring at the molecular level (1, 2). However, an in-depth biochemical characterization was limited for a long time by the rather low amount of protein available from expression in plant and insect cell cultures. Recently, we have developed an expression system in Pichia pastoris that enabled us to obtain sufficient quantities of purified BBE from Eschscholzia californica, allowing a more detailed characterization. In the course of these studies it was discovered that BBE belongs to a novel group of flavoproteins containing a bi-covalently attached flavin cofactor (3-7).In addition, sequence alignments reveal that several other proteins reported to possess an 8-␣-histidyl bond have the Cys residue involved in binding to position 6 of the cofactor conserved, e.g. ⌬ 1 -tetrahydrocannabinolic acid synthase from Cannabis sativa (8). Most of these enzymes with a confirmed or proposed bi-covalently attached flavin have a function in specialized metabolic pathways and catalyze reactions wit...

Identification of a Gatekeeper Residue That Prevents Dehydrogenases from Acting as Oxidases

“…The midpoint reduction potentials of wild type and A113G could be estimated from the log (E ox /E red ) versus log (dye ox /dye red ) plots and were Ϫ44 mV for wild-type GALDH and Ϫ63 mV for A113G. These values are in the same range as has been reported for other non-covalent flavoprotein dehydrogenases and oxidases (27), whereas covalent flavoproteins generally have higher reduction potentials (28,29). The Ala-113 to Gly mutation lowers the midpoint reduction potential of GALDH by about 20 mV.…”

A concerted mechanism for berberine bridge enzyme

“…The S N 2-type mechanism is in agreement with earlier studies demonstrating inversion of configuration at the N-methyl group 16 . The proposed hydride transfer to the flavin is in line with the absence of any flavin radical intermediate during substrate mediated reduction of the enzyme in stopped-flow experiments 17 (see also Supplementary Fig. 4 online).…”

Contribution of Flavin Covalent Linkage with Histidine 99 to the Reaction Catalyzed by Choline Oxidase

“…Glucooligosaccharide oxidase (15,16), hexose oxidase (17), and berberine bridge enzyme (18,19) are examples of flavoproteins (FAD as cofactor) with both linkages present in one flavin molecule. The covalent linkages in flavin-dependent enzymes have been shown to stabilize protein structure (20 -22), prevent loss of loosely bound flavin cofactors (23), modulate the redox potential of the flavin microenvironment (20,(23)(24)(25)(26)(27), facilitate electron transfer reactions (28), and contribute to substrate binding as in the case of the cysteinyl linkage (20). However, no study has implicated a mechanistic role of the flavin covalent linkages in enzymatic reactions in which a hydride ion is transferred by quantum mechanical tunneling.…”