Abstract
Flavin is a versatile cofactor involved in a wide spectrum of chemical transformations in biology. It is therefore not surprising that the flavoenzyme family represents one of the largest collections of redox enzyme molecules. The so-called covalent flavoproteins form a subgroup of this larger family of flavoproteins (1) and, although a relatively small group (approx 25 members as of 1998), covalent flavoproteins have received considerable attention, both from the viewpoint of enzyme mechanism and also the mode by which the flavin redox center becomes attached to the protein scaffold. The covalent flavoproteins fall into two categories—those in which the flavin is attached at the C6 position of the flavin isoalloxazine ring and those where linkage is via the 8α methyl group ( Fig. 1 ). The former group has only two members (tri- and dimethylamine dehydrogenases) and attachment occurs via a cysteine residue forming a C6-thioether linkage. The 8α methyl grouping forms the majority of the covalent flavoprotein family and members are typified by linkages between the 8α methyl group of the flavin isoalloxazine ring and histidine, tyrosine or cysteine side chains. The role of covalently bound flavins in redox enzymology has been the focus of much debate. For example, modulation of flavin reduction potential (2,3) and improvement of electron transfer rates to downstream redox acceptors by enhancing electronic coupling between cofactors (4) have been proposed. Recent work on trimethylamine dehydrogenase suggests that covalent linkage may have evolved to suppress hydroxylation of the isoalloxazine ring at the C6 position, thereby preventing inactivation of the redox center (5). Analysis of a variety of covalent flavoproteins has revealed that formation of the covalent link is a self-catalytic process (4,6,7), and mechanisms for flavinylation have been proposed. For selected enzymes, studies by directed mutagenesis have confirmed the roles of key residues in the flavinylation reaction [e.g., (4,8)].
Structures of the known flavin amino acids found in covalent flavoenzymes.
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References
Mewies, M., McIntire, W. S., and Scrutton, N. S. (1998) Covalent attachment of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to enzymes: the current state of affairs. Protein Sci. 7, 1–14.
Edmondson, D. E. and De Francisco, R. (1992) Structure, synthesis and physical properties of covalently bound flavins and 6-and 8-hydroxyflavins, in Chemistry and Biochemistry of Flavoenzymes, vol. 1 (Muller, F., ed.), Boca Raton, FL, CRC Press, pp. 73–103.
Williamson, G. and Edmondson, D. E. (1985) Effects of pH on oxidation-reduction potentials of 8α-N-substituted flavins. Biochemistry 24, 7790–7797.
Kim, J., Fuller, J. H., Kuusk, V., Cunane, L., Chen, Z., Mathews, F. S., and McIntire, W. S. (1995) The cytochrome subunit is necessary for covalent FAD attachment to the flavoprotein subunit of p-cresol methylhydroxylase. J. Biol. Chem. 263, 31,202–31,209.
Mewies, M., Basran, J., Packman, L. C., Hille, R., and Scrutton, N. S. (1997) Involvement of a flavin iminoquinone methide in the formation of 6-hydroxy FMN in trimethylamine dehydrogenase: a rationale for the existence of 8α-methyl and C6-linked covalent flavoproteins. Biochemistry 36, 7162–7168.
Scrutton, N. S., Packman, L. C., Mathews, F. S., Rohlfs, R. J., and Hille, R. (1994) Assembly of redox centers in the trimethylamine dehydrogenase of bacterium W3A1: properties of the wild-type enzyme and a C30A mutant expressed from a cloned gene in Escherichia coli. J. Biol. Chem. 269, 13,942–13,950.
Nagursky, H., Bichler, V., and Brandsch, R. (1988) Phosphoenolpyruvate-dependent flavinylation of 6-hydroxy-d-nicotine oxidase. Eur. J. Biochem. 177, 319–325.
Packman, L. C., Mewies, M., and Scrutton, N. S. (1995) The flavinylation reaction of trimethylamine dehydrogenase: analysis by directed mutagenesis and electrospray mass spectrometry. J. Biol. Chem. 270, 13,186–13,191.
Ohishi, N. and Yagi, K. (1979) Covalently bound flavin as prosthetic group of choline oxidase. Biochem. Biophys. Res. Comm. 86, 1084–1088.
Kiuchi, K., Nishikimi, M., and Yagi, K. (1982) Purification and characterisation of L-gulonolactone oxidase from chicken kidney microsomes. Biochemistry 21, 5076–5082.
Kutchan, T. M. and Dittrich, H. (1995) Characterisation and mechanism of the berberine bridge enzyme, a covalently flavinylated oxidase of benzophenanthridine alkaloid biosynthesis in plants. J. Biol. Chem. 270, 24,475–24,481.
Singer, T. P. and McIntire, W. S. (1984) Structure, properties and determination of covalently bound flavins. Methods Enzymol. 66, 253–264.
Kenney, W. C., McIntire, W. S., and Yamanaka, T. (1977) Structure of the covalently bound flavin of Chlorobium cytochrome c 553. Biochim. Biophys. Acta 483, 467–474.
Bruhmuller, M., and Decker, K. (1973) Covalently bound flavin in D-6-hydroxynicotine oxidase from Arthrobacter oxidans. Eur. J. Biochem. 37, 256–258.
Kenney, W. C., Edmondson, D. E., and Seng, R. L. (1976) Identification of the covalently bound flavin of thiamin dehydrogenase. J. Biol. Chem. 251, 5386–5390.
Kenney, W. C., Edmondson, D. E., Singer, T. P., Nishikimi, M., Noguchi, E., and Yagi, K. (1979) Identification of the covalently-bound flavin of L-galactonolactone oxidase from yeast. FEBS Lett. 97, 40–42.
Kenney, W. C., Singer, T. P., Fukuyama, M., and Miyake, Y. (1979) Identification of the covalently bound flavin prosthetic group of cholesterol oxidase. J. Biol. Chem. 254, 4689–4690.
Steenkamp, D. J., Kenney, W. C., and Singer, T. P. (1978) A novel type of covalently bound coenzyme in trimethylamine dehydrogenase. J. Biol. Chem. 253, 2812–2817.
McIntire, W., Edmondson, D. E., Singer, T. P., and Hopper, D. J. (1980) 8α-O-tyrosyl FAD: a new form of covalently bound flavin from p-cresol methylhydroxylase. J. Biol. Chem. 255, 6553–6555.
Kvalnes-Krick, K. and Jorns, M. S. (1986) Bacterial sarcosine oxidase: comparison of two multisubunit enzymes containing both covalent and noncovalent flavin. Biochemistry 25, 6061–6069.
De Jong, E., van Berkel, W. J. H., van der Zwan, R. P., and de Bont, J. A. M. (1992) Purification and characterisation of vanillyl-alcohol oxidase from Penicillium simplicissium. Eur. J. Biochem. 208, 651–657.
Walker, W. H., Kearney, E. B., Seng, R. L., and Singer, T. P. (1971) The covalently-bound flavin of hepatic monoamine oxidase: identification and properties of cysteinyl riboflavin. Eur. J. Biochem. 24, 328–331.
Edmondson, D. E., Kenney, W. C., and Singer, T. P. (1976) Structural elucidation and properties of 8α-(N 1-histidyl) riboflavin: the flavin component of thiamine dehydrogenase and cyclopiazonate oxidocyclase. Biochemistry 15, 2937–2945.
McIntire, W., Edmondson, D. E., Hopper, D. J., and Singer, T. P. (1981) 8α-(O-Tyrosyl)flavin adenine dinucleotide, the prosthetic group of bacterial p-cresol methylhydroxylase. Biochemistry 20, 3068–3075.
Steenkamp, D. J., McIntire, W., and Kenney, W. C. (1978). Structure of the covalently bound coenzyme of trimethylamine dehydrogenase: evidence for a 6-substituted flavin. J. Biol. Chem. 253, 2818–2824.
Singer, T. P., and Edmondson, D. E. (1980) Structure, properties, and determination of covalently bound flavins. Methods Enzymol. 66, 253–264.
Salach, J., Walker, W. H., Singer, T. P., Ehrenberg, A., Hemmerich, P., Ghisla, S., and Hartman, U. (1972) Studies on succinate dehydrogenase: site of attachment of the covalently-bound flavin to the peptide chain. Eur. J. Biochem. 26, 267–278.
Kenney, W. C. and Walker, W. H. (1972) Synthesis and properties of 8α-substituted riboflavins of biological importance. FEBS Lett. 20, 297–301.
Walker, W. H., Singer, T. P., Ghisla, S., and Hemmerich, P. (1972) Studies on succinate dehydrogenase: 8α-histidyl FAD as the active center of succinate dehydrogenase. Eur. J. Biochem. 26, 279–289.
Edmondson, D. E. (1976) 2′,5′-Anhydro-8α-histidyl flavins: their occurrence and structural elucidation. Fed. Proc. 35, 1542.
Kearney, E. B., Salach, J. I., Walker, W. H., Seng, R. L., Kenney, W., Zeszotek, E., and Singer, T. P. (1971) The covalently-bound flavin of hepatic monoamine oxidase. 1. Isolation and sequence of a flavin peptide and evidence for binding at the 8α position. Eur. J. Biochem. 24, 321–327.
Edmondson, D. E., Kenney, W. C., and Singer, T. P. (1978) Synthesis and isolation of 8α-substituted flavins and flavin peptides. Methods Enzymol. 53, 449–465.
Ghisla, S., Kenney, W. C., Knappe, W. R., McIntire, W. S., and Singer, T. P. (1980) Chemical synthesis and some properties of 6-substituted flavins. Biochemistry 19, 2537–2544.
Kenny, W. C. and Singer, T. P. (1977) Evidence for a thioether linkage between the flavin and polypeptide chain of Chromatium cyctochrome c 552. J. Biol. Chem. 252, 4767–4772.
Falk, M. C. and McCormick, D. B. (1976) Synthetic flavinyl peptides related to the active site of mitochondrial monoamine oxidase II. Fluorescence properties. Biochemistry 15, 646–653.
Perrett, D., Bhuste, L., Patel, J., and Herbert, K. (1991) Comparative performance of ion-exchange and ion-paired reversed phase high-performance liquid-chromatography for the determination of nucleotides in biological samples. Biomed. Chromatogr. 5, 207–211.
Randerath, K. and Randerath, E. (1967) Thin-layer separation methods for nucleic acid derivatives. Methods Enzymol. 12, 323–347.
Kenney, W. C., Edmondson, D. E., Singer, T. P., Steenkamp, D. J., and Schabort, J. C. (1976) Identification and properties of the covalently bound flavin of β-cyclopiazonate oxidocyclase. Biochemistry 15, 4931–4935.
McIntire, W. S., Singer, T. P., Ameyama, M., Adachi, O., Matsushita, K., and Shinagawa, E. (1985) Identification of the covalently-bound flavins of D-gluconate dehydrogenase from Pseudomonas aeroginosa and Pseudomonas fluorescens and of 2-keto-D-gluconate dehydrogenase from Gluconobacter melanogenus. Biochem. J. 231, 651–654.
Smith, I. (1960) Thin layer chromatography, in Chromatographic and Electrophoretic Techniques, vol. 1 (Smith, I., ed.), 2nd ed., Interscience, New York, pp. 66–103.
Koziol, J. (1971) Fluorometric analyses of riboflavin and its coenzymes. Methods Enzymol. 18B, 253–285.
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Scrutton, N.S. (1999). Identification of Covalent Flavoproteins and Analysis of the Covalent Link. In: Chapman, S.K., Reid, G.A. (eds) Flavoprotein Protocols. Methods in Molecular Biology, vol 131. Humana Press. https://doi.org/10.1385/1-59259-266-X:181
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DOI: https://doi.org/10.1385/1-59259-266-X:181
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