Structural and Mechanistic Studies Reveal the Functional Role of Bicovalent Flavinylation in Berberine Bridge Enzyme

Abstract

Berberine bridge enzyme (BBE) is a member of the recently discovered family of bicovalently flavinylated proteins. In this group of enzymes, the FAD cofactor is linked via its 8␣-methyl group and the C-6 atom to conserved histidine and cysteine residues, His-104 and Cys-166 for BBE, respectively. 6-S-Cysteinylation has recently been shown to have a significant influence on the redox potential of the flavin cofactor; however, 8␣-histidylation evaded a closer characterization due to extremely low expression levels upon substitution. Co-overexpression of protein disulfide isomerase improved expression levels and allowed isolation and purification of the H104A protein variant. To gain more insight into the functional role of the unusual dual mode of cofactor attachment, we solved the x-ray crystal structures of two mutant proteins, H104A and C166A BBE, each lacking one of the covalent linkages. Information from a structure of wild type enzyme in complex with the product of the catalyzed reaction is combined with the kinetic and structural characterization of the protein variants to demonstrate the importance of the bicovalent linkage for substrate binding and efficient oxidation. In addition, the redox potential of the flavin cofactor is enhanced additively by the dual mode of cofactor attachment. The reduced level of expression for the H104A mutant protein and the difficulty of isolating even small amounts of the protein variant with both linkages removed (H104A-C166A) also points toward a possible role of covalent flavinylation during protein folding.Since the discovery of the first known example of a covalent bond between a flavin cofactor and an amino acid side chain occurring in enzymes in the 1950s (1), a number of different types of linkages have been identified: 8␣-histidylation (either to N1 or to N3), 8␣-O-tyrosylation, 8␣-S-cysteinylation, and 6-S-cysteinylation. For current reviews relating to these modes of flavin attachment, see Refs. 2 and 3. Recently, another way of covalent tethering of FAD to proteins was discovered in x-ray crystallographic studies on glucooligosaccharide oxidase (GOOX) 4 from Acremonium strictum (4). The mode of flavin linkage observed in this case employs both 8␣-histidylation and 6-S-cysteinylation to form a bicovalently attached cofactor. Representative members of all these groups have been studied in detail, and several explanations for the role of the covalent flavinylation have been put forward. Some of the suggestions tend to be rather specific for the system being studied, e.g. prevention of cofactor inactivation at the C-6 position for trimethylamine dehydrogenase (5) or facilitation of electron transfer from the flavin to the cytochrome subunit for p-cresol methylhydroxylase (6). Other explanations including the increase of the flavin redox potential due to the covalent linkage (7-9) and the prevention of cofactor dissociation (10, 11) were found for several enzymes also harboring different types of cofactor attachments. Taking into account that protein stability (12)...

Oxidative Cyclization in Natural Product Biosynthesis

“…34,35,308–315 The enzyme converts ( S )-reticuline ( 118 ) derived from L-tyrosine, to ( S )-scoulerine ( 443 ) which is on the way to more complex benzophenanthridine alkaloids including berberine ( 205 ). BBE constructs a new C-C bond between the N- methyl and the catechol ring in 443 as shown in Scheme 61.…”

Biocatalytic Enantioselective Oxidative CC Coupling by Aerobic CH Activation

“…[11,12] Only recently, BBE from Eschscholzia californica (California poppy) could be expressed efficiently in Pichia pastoris [13] to give a sufficient quantity of the enzyme for crystallization and investigation of the mechanism. [10,14] Thus, BBE has been thoroughly investigated regarding its biochemical properties and catalytic mechanism, while its potential as a biocatalyst for preparative transformation of non-natural substrates has not been explored yet. A major concern referred to the question whether non-natural substrates could be transformed at all, since plant enzymes may have strict substrate specificity.…”

Biocatalytic Oxidative CC Bond Formation Catalysed by the Berberine Bridge Enzyme: Optimal Reaction Conditions

“…The enzyme contains a bicovalently bonded flavin responsible for the reaction. [21] Recently, it could be shown that recombinant BBE from Eschscholzia californica (California poppy) [22,23] can be employed efficiently for the preparation of novel optically pure isoquinoline and berbine alkaloids via enantioselective oxidative C À C bond formation.[24] The enzyme displayed perfect enantioselectiviScheme 1. Natural reaction catalysed by the berberine bridge enzyme (BBE).…”