Characterisation of wild‐type and mutant forms of human monoamine oxidase A and B expressed in a mammalian cell line

Gottowik, J.; Cesura, Andrea M.; Malherbe, P.; Lang, Gabrielle E.; Prada, M. Da

doi:10.1016/0014-5793(93)81512-x

Cited by 48 publications

(34 citation statements)

References 17 publications

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“…Each contains an FAD molecule covalently linked by an 8a-S-cysteinyl bond (Keamey et al, 1971;Walker et al, 1971;Nagy & Salach, 1981;Yu, 1981); the cofactor is linked to Cys 406 and Cys 397 in the A and B forms, respectively. Site-directed mutagenesis studies on MA0 B have shown that removal of the covalent link by replacing Cys 397 with Ser or His eliminates activity (Gottowik et al, 1993;Wu et al, 1993). Likewise, replacement of Cys 406 in the A form results in complete loss of catalytic activity (Wu et al, 1993).…”

Section: Monoamine Oxidase (La-s-cysteinyl 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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Section: Monoamine Oxidase (La-s-cysteinyl 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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“…They are closely linked on the X chromosome (Lan et al, 1989) and have an identical intron-exon organization, indicating that they are derived from a common ancestral gene (Grimsby et al, 1991). Knocking out the MAO A gene resulted in aggressive behavior in mice (Cases et al, 1995), whereas knocking out the MAO B gene resulted in mice resistant to the parkinsonism-inducing toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (Grimsby et al, 1997).To determine the region(s) responsible for the distinct substrate and inhibitor preferences of the two isoenzymes, we and other groups have made point mutants and chimeric MAO constructs exchanging corresponding portions of the isoenzymes (Gottowik et al, 1993(Gottowik et al, , 1995Wu et al, 1993;Tsugeno et al, 1995;Cesura et al, 1996;Chen et al, 1996). It has been shown that amino acid segments 161-375 in human MAO A and 152-366 in human MAO B contain part of the domain responsible for determining preference (Grimsby et al, 1996).…”

mentioning

confidence: 99%

“…To determine the region(s) responsible for the distinct substrate and inhibitor preferences of the two isoenzymes, we and other groups have made point mutants and chimeric MAO constructs exchanging corresponding portions of the isoenzymes (Gottowik et al, 1993(Gottowik et al, , 1995Wu et al, 1993;Tsugeno et al, 1995;Cesura et al, 1996;Chen et al, 1996). It has been shown that amino acid segments 161-375 in human MAO A and 152-366 in human MAO B contain part of the domain responsible for determining preference (Grimsby et al, 1996).…”

mentioning

confidence: 99%

Phe²⁰⁸ and Ile¹⁹⁹ in Human Monoamine Oxidase A and B Do Not Determine Substrate and Inhibitor Specificities as in Rat

Geha

Chen

Shih

2000

Journal of Neurochemistry

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It has been reported previously that reciprocally switching Phe 208 and Ile 199 in rat monoamine oxidase (MAO) A and B, respectively, was sufficient to switch their substrate and inhibitor preferences. In this study, the same mutants were made in the human forms of MAO. When compared with MAO A, MAO A-F208I showed a sixfold decrease in the specificity constant k cat /K m for both the MAO A-and the MAO B-preferring substrates 5-hydroxytryptamine and ␤-phenylethylamine, respectively. The reciprocal point mutant MAO B-I199F had no effect on substrate affinity. To investigate if the region neighboring these two residues is responsible for conferring preferences, we have also made chimeric constructs by reciprocally switching the corresponding amino acid segments 159 -214 in 1304 -1309 (2000).Monoamine oxidase [MAO; amine:oxygen oxidoreductase (deaminating) (flavin-containing), EC 1.4.3.4] catalyzes the oxidative deamination of biogenic and xenobiotic amines and plays an important role in regulating their levels. It is a flavin-adenine dinucleotidecontaining enzyme located on the mitochondrial outer membrane (Nara et al., 1966;Greenawalt and Schnaitman, 1970;Shih et al., 1999). It exists as two isoenzymes, A and B. MAO A preferentially oxidizes serotonin [5-hydroxytryptamine (5-HT)] and is inhibited by low concentrations of clorgyline, whereas MAO B preferentially oxidizes low concentrations of ␤-phenylethylamine (PEA) and is inhibited by low concentrations of deprenyl. Common substrates include tyramine (Johnston, 1968;Knoll and Magyar, 1972) and dopamine. MAO A and B are composed of 527 and 520 amino acids, respectively, and have a 70% amino acid identity (Bach et al., 1988). They are closely linked on the X chromosome (Lan et al., 1989) and have an identical intron-exon organization, indicating that they are derived from a common ancestral gene (Grimsby et al., 1991). Knocking out the MAO A gene resulted in aggressive behavior in mice (Cases et al., 1995), whereas knocking out the MAO B gene resulted in mice resistant to the parkinsonism-inducing toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (Grimsby et al., 1997).To determine the region(s) responsible for the distinct substrate and inhibitor preferences of the two isoenzymes, we and other groups have made point mutants and chimeric MAO constructs exchanging corresponding portions of the isoenzymes (Gottowik et al., 1993(Gottowik et al., , 1995Wu et al., 1993;Tsugeno et al., 1995;Cesura et al., 1996;Chen et al., 1996). It has been shown that amino acid segments 161-375 in human MAO A and 152-366 in human MAO B contain part of the domain responsible for determining preference (Grimsby et al., 1996). It was later discovered that reciprocally switching amino acids F208 in rat MAO A and its corresponding residue in rat MAO B, I199, was sufficient to switch the substrate and inhibitor preferences of rat MAO A and B (Tsugeno and Ito, 1997). To determine if the human MAO equivalents of these two residues, which are located within the segments in our ea...

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“…For transfection, HEK-293 cells (human embryonic kidney cells, ATCC CRL 1573) were seeded at a concentration of 2.5X105 celldm1 and grown for 24 h at 37°C under 5% CO,, in MEM+ (Gibco-BRL) with the addition of 20 mM Hepes, 10% fetal calf serum, 2 mM L-glutamine, 100 IU/ml penicillin and 100 pg/ml streptomycin. The subconfluent cells were washed with Dulbecco modified Eagle medium (Gibco-BRL) and incubated in the same medium with a mixture of 1 pg expression vector and 5 pg Transfectamm (IBF Biotechnics)/ml, for 2 h at 37 "C under 5 % CO, [15,331. The cells were grown for another 48 h in MEM+, then collected, washed twice with 20mM KH,PO, in 0.9% NaCI, pH 7.4, and frozen as a cell pellet.…”

Section: Engineering Of Mao-b Mutations a 1670-bp Mao-bmentioning

confidence: 99%

“…The selective MAO-A inhibitor Ro 41-1049 (2 pM) [25] was added in order to inhibit the low levels of endogenous MAO-A present in HEK-293 cells (>0.5 nmol/g protein) [15]. For kinetic analysis, the cell homogenates were incubated in the presence of at least six different [14C]phenylethylamine concentrations (range 0.2-20 pM).…”

Section: Engineering Of Mao-b Mutations a 1670-bp Mao-bmentioning

confidence: 99%

Investigation on the Structure of the Active Site of Monoamine Oxidase‐B by Affinity Labeling with the Selective Inhibitor Lazabemide and by Site‐Directed Mutagenesis

Cesura

Gottowik

Lahm

et al. 1996

European Journal of Biochemistry

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The structural features of the active site of human monoamine oxidase El (MAO-B) were investigated by affinity labeling and site-directed mutagenesis. The pseudosubstrate inhibitor N-[2-aminoethyl1-5-chloro-2-pyridine carboxamide HCI (lazabemide) can be irreversibly linked to MAO-B by reduction of the enzyme-inhibitor complex with NaBH,CN. Analysis of the flavin spectrum of ['Hllazabemide-labeled human MAO-B indicated that insertion of the inhibitor did not occur into the isoalloxazine ring of FAD. After trypsin digestion and HPLC peptide mapping of the radiolabeled enzyme, two labeled peptides were observed. Sequence analysis showed that both peptides started at Val371 of human MAO-B. These results indicate that ['Hllazabemide is incorporated into the MAO-B peptide stretch containing the FADmodified Cys397. The function of putative active-site residues contained in this region was investigated by site-directed mutagenesis and expression of the mutant proteins in HEK-293 cells. Substitution of His382 of MAO-B with an Arg greatly reduced the enzymic activity, suggesting that this residue may represent a nucleophile relevant for the MAO-B catalytic mechanism. Whereas it has been shown that MAO-A and MAO-B are the products of separate genes [5-71 encoding proteins with a high degree of amino acid identity (72.6%) [5]. In both M A 0 forms, the co-factor-binding site appears to be constituted by at least the following two regions: a domain near the N-terminus constituting the non-covalent FAD co-factor-binding site which contains an ADP-binding sequence

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Characterisation of wild‐type and mutant forms of human monoamine oxidase A and B expressed in a mammalian cell line

Cited by 48 publications

References 17 publications

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

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

Phe²⁰⁸ and Ile¹⁹⁹ in Human Monoamine Oxidase A and B Do Not Determine Substrate and Inhibitor Specificities as in Rat

Investigation on the Structure of the Active Site of Monoamine Oxidase‐B by Affinity Labeling with the Selective Inhibitor Lazabemide and by Site‐Directed Mutagenesis

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Characterisation of wild‐type and mutant forms of human monoamine oxidase A and B expressed in a mammalian cell line

Cited by 48 publications

References 17 publications

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

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

Phe208 and Ile199 in Human Monoamine Oxidase A and B Do Not Determine Substrate and Inhibitor Specificities as in Rat

Investigation on the Structure of the Active Site of Monoamine Oxidase‐B by Affinity Labeling with the Selective Inhibitor Lazabemide and by Site‐Directed Mutagenesis

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Phe²⁰⁸ and Ile¹⁹⁹ in Human Monoamine Oxidase A and B Do Not Determine Substrate and Inhibitor Specificities as in Rat