Covalent cofactor binding to flavoenzymes requires specific effectors

Brandsch, Roderich; Bichler, Veronika

doi:10.1111/j.1432-1033.1989.tb14808.x

Cited by 43 publications

(27 citation statements)

References 20 publications

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“…In vitro flavinylation experiments with isolated Me,GlyDH, however, were thus far, unsuccessful. We have recently shown that in the bacterial enzyme 6-hydroxy-~-nicotine oxidase, the covalent incorporation of FAD is an autocatalytic process linked to a flavinylation-competent conformational state of the protein [27] (unpublished observations). Experiments with recombinant Me,GlyDH expressed and purified from E. coli are under way to investigate this possibility.…”

Section: Discussionmentioning

confidence: 92%

Rat liver dimethylglycine dehydrogenase

Lang

Polster

Brandsch

1991

European Journal of Biochemistry

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Dimethylglycine dehydrogenase (Me2GlyDH), an enzyme of choline catabolism specifically expressed in the mammalian liver, was analyzed in rat hepatocytes in culture. This mitochondrial enzyme carries the FAD cofactor covalently attached to the polypeptide chain by its riboflavin 8a position to N" of histidine [Cook, R., Misono, Biol. Chem. 256,4102-41081, and a 43-amino-acid leader peptide. The Nterminus of Me,GlyDH contains a conserved amino acid sequence which forms the dinucleotide-binding site in many enzymes with noncovalently bound FAD. Close to the modified histidine there is an amino acid sequence resembling a sequence conserved in thymidylate synthases and shown in these enzymes to be involved in the binding of the pteroyl polyglutamate cofactor.Choline, taken up with the food or set free from phosphatidylcholine, is degraded in mammals, specifically in the liver. The enzymes of this pathway are mitochondrially located. The first, choline dehydrogenase, situated on the matrix side of the inner mitochondrial membrane, contains FAD and has a function similar to the succinate dehydrogenase complex in relation to the respiratory chain [l]. Two additional flavoenzymes of the pathway, dimethylglycine dehydrogenase (Me,GlyDH) and sarcosine dehydrogenase, were shown to contain, besides tightly bound pteroyl pentaglutamate, FAD covalently attached to the polypeptide chain by its riboflavin 8a position to N" of histidine [2].We have investigated the biogenesis of this covalent modification in 6-hydroxy-~-nicotine oxidase, an enzyme of procaryotic origin. With this particular enzyme the histidyl His(FAD) linkage is synthesized in an autocatalytic process by the polypeptide itself. It is not known how this protein modification is accomplished in enzymes of the eucaryotic Correspondence to R. Brandsch, Biochemisches Institut, Universitat Freiburg, Hermann-Herder-Str. 7, W-7800 Freiburg, Federal Republic of Germany Abbreviations. His(FAD), FAD linked through riboflavin position 8r to N" of histidine; MeZGlyDH, dimethylglycine dehydrogenase.Enzyme. Dimethylglycine dehydrogenase (EC 1.5.99.2). Nore. The nucleotide sequence corresponding to the coding part of the sequence reported in this paper is available from the EMBL data bank with accession number X55995.cell. In the case of other cofactors, covalent attachment to the protein, like biotin in carboxylases or heme in cytochrome c, requires a special enzyme [3, 41. The attachment of the prosthetic group is a step involved in the import of apocytochrome c into the intermembrane space and in its correct location within the inner mitochondrial membrane [S]. Whether a similar mitochondrial factor is involved in the covalent attachment of FAD to flavoenzymes is not known. It would also be of interest to know at which stage in the pathway from the precursor protein to the mature form the covalent modification takes place and how it influences the folding of the polypeptide chain on its way to the mature, enzymatically active form.In order to address these questions, we have...

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Section: Discussionmentioning

confidence: 92%

Rat liver dimethylglycine dehydrogenase

Lang

Polster

Brandsch

1991

European Journal of Biochemistry

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“…Cultures were harvested, lysed by lysozymes (20 g/ml) and sonication (6 ϫ 10-s bursts) in 0.1 M NaPO 4 buffer (pH 7), and then centrifuged at 150,000 ϫ g for 30 min with the resulting supernatant being kept at 4°C for future use (19). FAD binding assays involved incubating 40 l of WT ϩ SdhA N-His (ϳ1 mg of total protein) or 83 l of ⌬sdhE ϩ SdhA N-His (ϳ2 mg of total protein) with 10 g of FAD with or without 35 g of purified SdhE N-His in a 100-l reaction buffered with 0.1 M NaPO 4 (pH 7).…”

Section: Methodsmentioning

confidence: 99%

SdhE Is a Conserved Protein Required for Flavinylation of Succinate Dehydrogenase in Bacteria

McNeil

Clulow

Wilf

et al. 2012

Journal of Biological Chemistry

120

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“…Full recovery of MAO B enzymatic activity obtained in Rib ϩ COS-7 cells was not achieved for reasons that remain unknown. In related studies, however, Brandsch and Bichler (33) found that the covalent flavinylation of 6-hydroxy-D-nicotine oxidase in vitro required specific effectors (phosphorylated three carbon compounds), such as glycerol 3-phosphate, glyceraldehyde 3-phosphate, or glycerate 3-phosphate. Effectors that could enhance the activity of MAO B have not been identified.…”

Section: Table I Comparison Of Mao B Enzymatic Activity and Mao B Expmentioning

confidence: 98%

Flavinylation of Monoamine Oxidase B

Zhou

Lewis

Kwan

et al. 1995

Journal of Biological Chemistry

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Monoamine oxidase B (MAO B)The major amine-degrading enzymes in the central nervous system and peripheral tissues of mammals are monoamine oxidase A and B (MAO 1 A and B, amine:oxygen, oxidoreductase (deaminating, flavin-containing), EC 1.4.3.4). These isozymes are integral proteins of the outer mitochondrial membrane (1) and can be distinguished by differences in substrate preference (2), inhibitory specificity (3), tissue and cell distribution (4 -6), and immunological properties (7-9). Furthermore, comparison of their nucleotide and deduced amino acid sequences show that human MAO A and B are two distinct proteins encoded by different genes (10).Oxidation of amines by MAO is coupled to the reduction of an obligatory cofactor, FAD, which is covalently linked to the enzyme. Five types of bonds are generally found in the covalent linkage of flavins to their respective apoproteins (11). These include a histidine residue which can be attached through its N-1 or N-3 atom to the 8␣-methyl group of the isoalloxazine ring to form a tertiary amine; a cysteine residue which forms a thioether linkage with either the 8␣-methyl group or the C-6 of the xylene ring of the flavin molecule; or a tyrosine residue can become linked to the 8␣-methyl group to form an (O)-8␣-flavin bond. In MAO A and B, the 8␣-methyl group of FAD is bound covalently to cysteine through a thioether linkage in the pentapeptide SGGCY (12, 13). Comparison of this segment with the complete deduced amino acid sequences of MAO A and B indicated that FAD is covalently bound to Cys 406 in MAO A and Cys 397 in MAO B, respectively (10). In addition, site-directed mutagenesis studies of MAO B, where Cys 397 was substituted with serine or histidine, showed that this cysteine residue is essential for catalytic activity (14,15).Although the amino acid sequences surrounding the FAD covalent attachment site in different flavoproteins bear little homology, a distinct non-covalent FAD-binding site displays high sequence identity in many FAD-containing enzymes of diverse function (16,17). This non-covalent FAD binding region is commonly referred to as the dinucleotide-binding site or motif due to its interaction with the AMP moiety of FAD. This motif consists of a ␤ 1 -sheet-␣-helix-␤ 2 -sheet beginning with a highly conserved Gly-X-Gly-X-X-Gly sequence between the first ␤-sheet and the ␣-helix. The second ␤-sheet usually ends with a glutamate residue in which the ␥-carboxylate group is thought to interact through a hydrogen bond with the 2Ј-hydroxyl group of ribose in the AMP moiety of FAD. In MAO A and B, this motif is located at the N terminus of MAO A (residues 15-43) and MAO B (residues 6 -34) and ends in Glu 43 and Glu 34 , respectively. Site-directed mutagenesis studies, where Glu 34 was replaced with aspartate, glutamine, or alanine, resulted in near complete or total loss of catalytic activity in MAO B (18).A fundamental process in the intracellular generation of functional flavoenzymes is the molecular mechanism which generates holoenzyme from apoenzyme a...

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Covalent cofactor binding to flavoenzymes requires specific effectors

Cited by 43 publications

References 20 publications

Rat liver dimethylglycine dehydrogenase

Rat liver dimethylglycine dehydrogenase

SdhE Is a Conserved Protein Required for Flavinylation of Succinate Dehydrogenase in Bacteria

Flavinylation of Monoamine Oxidase B

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