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

Abstract

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...

Oxygen Access to the Active Site of Cholesterol Oxidase through a Narrow Channel Is Gated by an Arg-Glu Pair

“…A number of flavoenzymes have been found to contain a covalent linkage between the cofactor and the protein (reviewed by Mewies et al (22)). The structures of covalently modified flavoenzymes that have been determined include VAO (17), where the covalent linkage is through ND2 of a histidine residue and p-cresol methylhydroxylase (20) where the linkage is through a tyrosine residue.…”

Expression inEscherichia coliof an Unnamed Protein Gene fromAspergillus oryzaeRIB40 and Cofactor Analyses of the Gene Product as Formate Oxidase

“…The extract showed a hypsochromic shift of the absorption band or the fluorescence band in the near-UV region, as compared with FAD, a characteristics of 8-substitude flavins. [12][13][14][15][16][17] 8 -(N-Histidyl)-FAD and peptides having covalent flavins with a histidyl linkage at the 8 position are reported to exhibit a characteristic increase in fluorescence below pH 7.0. 12,14,17) These results together with the presence of a conserved motif of the FAD binding site in the molecule of FOD AO suggest that FOD AO contains a non-covalently bound FAD analog with a modification on the isoalloxazine ring, and that the modified FAD is 8-substituted FAD.…”

YgfX (CptA) is a multimeric membrane protein that interacts with the succinate dehydrogenase assembly factor SdhE (YgfY)

“…The loss of Sdh5/SdhE prevents FAD incorporation into Sdh1/SdhA and results in a nonfunctional SDH (Hao et al, 2009;McNeil et al, 2012). The process of flavinylation has traditionally been thought of as autocatalytic (Edmondson & Newton-Vinson, 2001;Heuts et al, 2009;Kim et al, 1995;Mewies et al, 1998). Consequently, SdhE was the first FAD assembly factor identified in bacteria (McNeil et al, 2012).…”