Characterisation of wild-type and mutant forms of human monoamine oxidase A and B expressed in a mammalian cell line.
Monoamine oxidase (MAO)-A and MAO-B are FAD-containing mitochondrial enzymes which catabolize biogenic and xenobiotic amines. The N-terminal regions of both forms of MAO contain an ADP-binding consensus sequence found in several dinucleotide-dependent enzymes, but otherwise show remarkable sequence differences. In order to investigate whether the N-terminal region of MAOs participates in the different catalytic properties and inhibitor specificities exhibited by MAO-A and MAO-B, we constructed chimeric A/B forms and expressed them in a human embryonic kidney cell line (293 cells). The MAO-A chimeric form containing the N-terminus (36 amino acids) of MAO-B and the B chimera having the first 45 amino acid sequence of MAO-A were both catalytically active. Compared to the respective wild-type form, they did not show any significant difference in their catalytic properties (Km, kcat) towards the substrates tested or in their sensitivity towards inhibitors. This indicates that the N-terminal region of the two isoenzymes is not involved in the different specificities of MAO-A and MAO-B. Substitution of Cys-397 of MAO-B, i.e. the residue covalently anchoring FAD, with an Ala or a His residue resulted in the total loss of enzymatic activity, suggesting that the covalent coupling of FAD to MAO occurs specifically at the-SH group of cysteine.
Gottowik
J
J
Pharma Division, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Cesura
A M
AM
Malherbe
P
P
Lang
G
G
Da Prada
M
M
D002478:Cells, Cultured; D003545:Cysteine / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008995:Monoamine Oxidase / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D005182:Flavin-Adenine Dinucleotide; D008995:Monoamine Oxidase; D003545:Cysteine