A concerted mechanism for berberine bridge enzyme

Winkler, Andreas; Lyskowski, Andrzej Franciszek; Riedl, Sabrina; Puhl, Martin; Kutchan, Toni M.; Macheroux, Peter; Gruber, Karl

doi:10.1038/nchembio.123

Cited by 132 publications

(162 citation statements)

References 19 publications

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“…The three-dimensional x-ray crystal structures of BBE, glucooligosaccharide oxidase, 6-hydroxy-D-nicotine oxidase, and aclacinomycin oxidoreductase showed that the flavoproteins of the BBE subfamily comprise two domains: a conserved FAD-binding domain and an a/b-domain with a seven-stranded, antiparallel b-sheet forming the less-conserved substrate-binding domain. BBE (Winkler et al, 2008), glucooligosaccharide oxidase (Huang et al, 2005), and aclacinomycin oxidoreductase (Alexeev et al, 2007) bicovalently attach FAD to the protein via two amino acid residues, His and Cys, whereas 6-hydroxy-D-nicotine oxidase covalently binds the flavin cofactor only via a His residue (Koetter and Schulz, 2005). In BBLs, the His residue, which forms the covalent linkage of FAD in these four enzymes, is conserved, but the Cys residue, which is used for the bicovalent linkage, is missing, except for BBLd, whose expression was not detected in the tobacco roots (Fig.…”

Section: Discussionmentioning

confidence: 99%

Vacuole-Localized Berberine Bridge Enzyme-Like Proteins Are Required for a Late Step of Nicotine Biosynthesis in Tobacco1

et al. 2011

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Tobacco (Nicotiana tabacum) plants synthesize nicotine and related pyridine-type alkaloids, such as anatabine, in their roots and accumulate them in their aerial parts as chemical defenses against herbivores. Herbivory-induced jasmonate signaling activates structural genes for nicotine biosynthesis and transport by way of the NICOTINE (NIC) regulatory loci. The biosynthesis of tobacco alkaloids involves the condensation of an unidentified nicotinic acid-derived metabolite with the N-methylpyrrolinium cation or with itself, but the exact enzymatic reactions and enzymes involved remain unclear. Here, we report that jasmonate-inducible tobacco genes encoding flavin-containing oxidases of the berberine bridge enzyme family (BBLs) are expressed in the roots and regulated by the NIC loci. When expression of the BBL genes was suppressed in tobacco hairy roots or in tobacco plants, nicotine production was highly reduced, with a gradual accumulation of a novel nicotine metabolite, dihydromethanicotine. In the jasmonate-elicited cultured tobacco cells, suppression of BBL expression efficiently inhibited the formation of anatabine and other pyridine alkaloids. Subcellular fractionation and localization of green fluorescent protein-tagged BBLs showed that BBLs are localized in the vacuoles. These results indicate that BBLs are involved in a late oxidation step subsequent to the pyridine ring condensation reaction in the biosynthesis of tobacco alkaloids.

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Section: Discussionmentioning

confidence: 99%

Vacuole-Localized Berberine Bridge Enzyme-Like Proteins Are Required for a Late Step of Nicotine Biosynthesis in Tobacco1

et al. 2011

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“…About a tenth of all flavoproteins have been shown to contain a covalently attached cofactor, which may be linked at the C8M position via histidyl, tyrosyl, or cysteinyl side chains or at the C6M position via a cysteinyl side chain (14). Glucooligosaccharide oxidase (15, 16), hexose oxidase (17), and berberine bridge enzyme (18,19) The discovery of quantum mechanical tunneling in enzymatic reactions, in which hydrogen atoms, protons, and hydride ions are transferred, has attracted considerable interest in enzyme studies geared toward understanding the mechanisms underlying the several orders of magnitudes in the rate enhancements of protein-catalyzed reactions compared with non-enzymatic ones. Tunneling mechanisms have been shown in a wide array of cofactor-dependent enzymes, including flavoenzymes.…”

mentioning

confidence: 99%

“…About a tenth of all flavoproteins have been shown to contain a covalently attached cofactor, which may be linked at the C8M position via histidyl, tyrosyl, or cysteinyl side chains or at the C6M position via a cysteinyl side chain (14). 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).…”

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confidence: 99%

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

Quaye¹,

Cowins

Gadda

2009

Journal of Biological Chemistry

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A number of enzymes, including dehydrogenases (1-3), monooxygenases (4 -7), halogenases (8 -11), and oxidases (7, 12, 13), employ flavin cofactors (FAD or FMN) for their catalytic processes. About a tenth of all flavoproteins have been shown to contain a covalently attached cofactor, which may be linked at the C8M position via histidyl, tyrosyl, or cysteinyl side chains or at the C6M position via a cysteinyl side chain (14). Glucooligosaccharide oxidase (15, 16), hexose oxidase (17), and berberine bridge enzyme (18,19) The discovery of quantum mechanical tunneling in enzymatic reactions, in which hydrogen atoms, protons, and hydride ions are transferred, has attracted considerable interest in enzyme studies geared toward understanding the mechanisms underlying the several orders of magnitudes in the rate enhancements of protein-catalyzed reactions compared with non-enzymatic ones. Tunneling mechanisms have been shown in a wide array of cofactor-dependent enzymes, including flavoenzymes. Examples of flavoenzymes in which the tunneling mechanisms have been demonstrated include morphinone reductase (29, 30), pentaerythritol tetranitrate reductase (29), glucose oxidase (31-33), and choline oxidase (34). Mechanistic data on Class 2 dihydroorotate dehydrogenases, also with a flavin cofactor (FMN) covalently linked to the protein moiety (35,36), could only propose a mechanism that is either stepwise or concerted with significant quantum mechanical tunneling for the hydride transfer from C6 and the deprotonation at C5 in the oxidation of dihydroorotate to orotate (37). This leaves choline oxidase as the only characterized enzyme with a covalently attached flavin cofactor (12,38), where the oxidation of its substrate occurs unequivocally by quantum mechanical tunneling.Choline oxidase from Arthrobacter globiformis catalyzes the two-step FAD-dependent oxidation of the primary alcohol substrate choline to glycine betaine with betaine aldehyde, which is predominantly bound to the enzyme and forms a gem-diol species, as intermediate (Scheme 1). Glycine betaine accumulates in the cytoplasm of plants and bacteria as a defensive mechanism against stress conditions, thus making genetic engineering of relevant plants of economic interest (39 -45), and the biosynthetic pathway for the osmolyte is a potential drug target in human microbial infections of clinical interest (46 -48). The first oxidation step catalyzed by choline oxidase involves the transfer of a hydride ion from a deprotonated choline to the protein-bound flavin followed by reaction of the anionic flavin hydroquinone with molecular oxygen to regenerate the oxidized FAD (for a recent review see Ref. 50). The gem-diol choline, i.e. hydrated betaine aldehyde, is the substrate for the second oxidation step (49), suggesting that the reaction may follow a similar mechanism. The isoalloxazine ring of the flavin cofac-

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“…21 were used for seeding of crystallization setups with protein variants H104A and C166A. For this, drops of 1 l of enzyme solution (ϳ25 mg ml Ϫ1 in 50 mM Tris/HCl, 150 mM NaCl, pH 9.0) were mixed with 1 l of reservoir solution (0.2 M MgCl 2 and 30% (w/v) polyethylene glycol 4000 in 0.1 M Tris/HCl, pH 8.5) and equilibrated for 2 h prior to streak seeding.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, a structure of wild type (WT) BBE in complex with the product of the enzyme-catalyzed reaction is presented, which provides further insights toward the involvement of active site amino acids during the course of the reaction. Together with the recently reported x-ray crystal structure of WT BBE with and without substrate bound (21) and the biochemical characterization of the C166A mutant protein (20), these results provide interesting insights into the role of bicovalent FAD attachment in enzymes.…”

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confidence: 99%

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

Winkler

Motz

Riedl

et al. 2009

Journal of Biological Chemistry

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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)...

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A concerted mechanism for berberine bridge enzyme

Cited by 132 publications

References 19 publications

Vacuole-Localized Berberine Bridge Enzyme-Like Proteins Are Required for a Late Step of Nicotine Biosynthesis in Tobacco1

Vacuole-Localized Berberine Bridge Enzyme-Like Proteins Are Required for a Late Step of Nicotine Biosynthesis in Tobacco1

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

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

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