Chemical synthesis and some properties of 6-substituted flavins

Ghisla, Sandro; Kenney, William C.; Knappe, Wolfgang‐R.; McIntire, William S.; Singer, Thomas P.

doi:10.1021/bi00553a001

Cited by 42 publications

(43 citation statements)

References 27 publications

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“…Therefore, our data clearly show that also for bi-covalently flavinylated proteins the modification in position 6 of the isoalloxazine ring system significantly increases the midpoint potential by a value of ϩ79 mV. The effect is higher than expected from studies with free FMN and its 6-cysteinylated analogue (⌬E M ϭ 57 mV) (34). To the best of our knowledge, the flavoproteins with the highest potentials reported so far all belong to the p-cresol methylhydroxylase superfamily and have midpoint potentials up to 55 mV for vannilyl alcohol oxidase (11) and 84 mV in the case of p-cresol methylhydroxylase (35).…”

Section: Discussionsupporting

confidence: 46%

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

Winkler

Kutchan

Macheroux

2007

Journal of Biological Chemistry

101

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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...

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Section: Discussionsupporting

confidence: 46%

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

Winkler

Kutchan

Macheroux

2007

Journal of Biological Chemistry

101

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“…These flavins and some of their properties had previously been reported in connection with the structural determination of the covalently bound flavin of trimethylamine dehydrogenase [50,51]. They promise to be very useful for probing the active site environment around the flavin 6-position, a position of interest because of its close proximity to the redox-active 5-position.…”

Section: Chemically Reactive Probesmentioning

confidence: 98%

New flavins for old: artificial flavins as active site probes of flavoproteins

Ghisla¹,

Massey²

1986

Biochemical Journal

195

143

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Section: Wiethylamine Dehydrogenase (6-s-cysteinyl Fmn)mentioning

confidence: 99%

“…At low pH, 6-S-cysteinyl riboflavin gives rise to a blue fluorescent photoproduct and, at high pH, a nonfluorescent photoproduct. Because routes to the synthesis of Sa-aminoacyl flavins (Edmondson et al, 1978;Singer & Edmondson, 1980; McIntire et al, 1981) and 6-S-cysteinyl riboflavin (Ghisla et al, 1980) are known, unequivocal identification is made by comparing the mobility of the unknown sample with synthetic standards during, e.g., TLC, HPLC, or by mass analysis. …”

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confidence: 99%

“…They are the first and only enzymes discovered to contain covalently bound flavin with substitution in other than the Sa position. Evidence for substitution at the C6 position was gained from UVvisible spectroscopy of a flavinylated peptide (Steenkamp & Singer, 1976;Steenkamp et al, 1978), NMR data, comparison of spectroscopic data with 6-hydroxy flavins and reaction of N-ethylmaleimide with the product of anaerobic photolysis of the coenzyme , and comparison with synthetic 6-S-cysteinylriboflavin (Ghisla et al, 1980).The mechanism of formation of the covalent link has been studied with TMADH. Expression of the cloned gene (Boyd et al, 1992) in the heterologous host E. coli leads to partial flavinylation of the enzyme (Scrutton et al, 1994;Packman et al, 1995), suggesting that formation of the 6-S-cysteinyl FMN link is selfcatalytic.…”

mentioning

confidence: 99%

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Covalent attachment of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to enzymes: The current state of affairs

1998

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The first identified covalent flavoprotein, a component of mammalian succinate dehydrogenase, was reported 42 years ago. Since that time, more than 20 covalent flavoenzymes have been described, each possessing one of five modes of FAD or FMN linkage to protein. Despite the early identification of covalent flavoproteins, the mechanisms of covalent bond formation and the roles of the covalent links are only recently being appreciated. The main focus of this review is, therefore, one of mechanism and function, in addition to surveying the types of linkage observed and the methods employed for their identification. Case studies are presented for a variety of covalent flavoenzymes, from which general findings are beginning to emerge.Keywords: covalent flavoproteins; flavin adenine dinucleotide; flavin mononucleotide; flavinylation; redox cofactors Cofactors are recruited by protein molecules to extend the chemistry available within the active sites of enzymes. The chemistries displayed are variable and the range of protein-specific cofactors available bears testimony to the types of biological reaction achieved by many enzyme molecules. Cofactors may be present in the freely dissociable form, but others, such as lipoic acid and biotin, are permanently linked to the host protein. Other cofactors (e.g., heme and pyridoxyl phosphate) fall into both categories in that they can be attached covalently to the protein or operate in the freely dissociable form. The covalent addition of specific cofactors to unique residues within a polypeptide chain is a fundamental problem in studies of biological molecular recognition. In many cases, this specificity is purchased at the expense of recruiting modification enzymes that direct the covalent addition of a cofactor to the host protein. For example, the additions of biotin and lipoic acid to the €-amino groups of specific Lys residues in carboxyltransferases and the 2-oxoacid dehydrogenases are directed by a biotin holoenzyme synthase (Sweetman et al., 1985;Takai et al., 1987) and Reprint requests to: N.S. Scmtton, Department of Biochemistry, University of Leicester, Adrian Building, University Road, Leicester LE1 7RH UK; e-mail: nss4@le.ac.uk; or W.S. McIntire, Molecular Biology Division, Department of Veterans Affairs Medical Center, San Francisco, California 94121; e-mail: wsm@itsa.ucsf.edu.lipoate-protein ligases (Brookfield et al., 1991; Moms et al., 1994), respectively. In a similar fashion, a heme cytochrome c lyase (Steiner et al., 1996) is responsible for the covalent addition of heme to two Cys residues via thioether linkages. In some enzymes, an existing side chain is modified to yield what is in effect a covalently attached cofactor, although no pre-existing cofactor molecule is incorporated into the enzyme. Several examples of this type of modification are provided in the next paragraph.For histidine decarboxylase of Lactobacillus 30A (Recsei & Snell, 1984), a pyruvoyl group is generated from an existing residue, Ser 82. The enzyme is synthesized as a proenzym...

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Chemical synthesis and some properties of 6-substituted flavins

Cited by 42 publications

References 27 publications

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

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

New flavins for old: artificial flavins as active site probes of flavoproteins

Covalent attachment of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to enzymes: The current state of affairs

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