The design of an alternative, covalently flavinylated 6‐hydroxy‐<scp>d</scp>‐nicotine oxidase by replacing the FAD‐binding histidine by cysteine and reconstitution of the holoenzyme with 8‐(methylsulfonyl)FAD

Stoltz, Michaela; Henninger, Hanspeter; Brandsch, Roderich

doi:10.1016/0014-5793(96)00438-3

Cited by 12 publications

(11 citation statements)

References 16 publications

(17 reference statements)

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“…Because of this specificity, the extra-histidines of the tag are very unlikely to interact in any way with the FAD moiety. Consistently, a number of different His-tagged apo-flavoproteins have been generated in other laboratories: they all bind FAD correctly [30,[44][45][46]. Moreover, in our flavinylation assay, the mature His-tagged holoenzyme does not incorporate additional FAD molecules (data not shown).…”

Section: Autoflavinylation Of Apo-pme 2 Glydh-hissupporting

confidence: 77%

The purified recombinant precursor of rat mitochondrial dimethylglycine dehydrogenase binds FAD via an autocatalytic reaction

Brizio¹,

Brandsch

Douka³

et al. 2008

International Journal of Biological Macromolecules

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Section: Autoflavinylation Of Apo-pme 2 Glydh-hissupporting

confidence: 77%

The purified recombinant precursor of rat mitochondrial dimethylglycine dehydrogenase binds FAD via an autocatalytic reaction

Brizio¹,

Brandsch

Douka³

et al. 2008

International Journal of Biological Macromolecules

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“…Their reconstitution with native and artificial cofactors has been used to investigate how the covalent linkage between the methyl group of the isoalloxazine ring and the protein residues is formed and whether it is important for the catalytic activity of the protein. [29][30][31][32][33] In a recent example, large amounts of soluble apoenzyme of monomeric sarcosine oxidase (MSOX) were produced by controlled expression in a riboflavin-dependent Escherichia coli strain. Its reconstitution with native flavin led to about 80 % restoration of its native activity and to spectroscopic and catalytic properties indistinguishable from those of the native MSOX containing covalently bound flavin.…”

Section: Flavin Reconstitution In Structural and Catalytic Studiesmentioning

confidence: 99%

Apoenzyme Reconstitution as a Chemical Tool for Structural Enzymology and Biotechnology

et al. 2009

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Many enzymes contain a nondiffusible organic cofactor, often termed a prosthetic group, which is located in the active site and essential for the catalytic activity of the enzyme. These cofactors can often be extracted from the protein to yield the respective apoenzyme, which can subsequently be reconstituted with an artificial analogue of the native cofactor. Nowadays a large variety of synthetic cofactors can be used for the reconstitution of apoenzymes and, thus, generate novel semisynthetic enzymes. This approach has been refined over the past decades to become a versatile tool of structural enzymology to elucidate structure-function relationships of enzymes. Moreover, the reconstitution of apoenzymes can also be used to generate enzymes possessing enhanced or even entirely new functionality. This Review gives an overview on historical developments and the current state-of-the-art on apoenzyme reconstitution.

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“…Recent studies have focused on the requirement for flavinylation in the mitochondrial import of 6HDNO-dimethylglycine dehydrogenase fusions (Stoltz et al, 1995), the interaction of 6HDNO with the chaperone protein GroE (Brandsch et al, 1992), and the covalent incorporation of flavin analogues (modified in the adenine moiety of FAD) into the active site of the enzyme (Stoltz et al, 1996a(Stoltz et al, , 1996b. In oxidase, where the FAD-linking His residue was replaced with Cys, noncovalently bound FAD could be displaced rapidly by added 8-methylsulfonyl-FAD or 8-chloro-FAD (Stolz et al, 1996b).…”

Section: -Hydroxy-~-nicotine Oxidase (Su-n3-histidyl Fad)mentioning

confidence: 99%

“…Either becomes covalently attached to the substitute Cys to produce the covalent 8-S-cysteinyl-FAD, not Sa-S-cysteinyl-FAD as in the normal enzyme. [Note: The 8-S-cysteinyl-FAD bound to the mutant form of 6HDNO was misidentified as 8-N-acetylcysteinyl-FAD by Stoltz et al (1996a). The latter is the synthetic model compound used for comparison of physical properties (see Massey et al, 1979, as quoted by Stoltz et a].).…”

Section: -Hydroxy-~-nicotine Oxidase (Su-n3-histidyl Fad)mentioning

confidence: 99%

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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The design of an alternative, covalently flavinylated 6‐hydroxy‐d‐nicotine oxidase by replacing the FAD‐binding histidine by cysteine and reconstitution of the holoenzyme with 8‐(methylsulfonyl)FAD

Cited by 12 publications

References 16 publications

The purified recombinant precursor of rat mitochondrial dimethylglycine dehydrogenase binds FAD via an autocatalytic reaction

The purified recombinant precursor of rat mitochondrial dimethylglycine dehydrogenase binds FAD via an autocatalytic reaction

Apoenzyme Reconstitution as a Chemical Tool for Structural Enzymology and Biotechnology

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

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