Structure/function relationships of mitochondrial monoamine oxidase A and B chimeric forms.
Monoamine oxidases (MAO) A and B show a high degree of amino acid similarity. Apart from the NH2-terminus, which contains an ADP-binding consensus sequence, little is known about their structural features or the sequences involved in the binding of substrates. In the present paper, we have studied the structure/function relationships of MAOs by constructing 18 different chimeric forms of MAO, engineered by moving progressively the junction between the NH2-terminus of one MAO form with the COOH-terminus of its isoenzyme. After transient expression in HEK-293 cells, the properties of these chimeric enzymes were investigated using both selective and nonselective substrates and inhibitors. Whereas exchange of the ADP-binding sequence did not modify the catalytic properties of either MAO isoforms, chimeras with increasing length of the NH2-terminus of MAO-A (up to residue 256) showed a marked decrease in affinity towards the MAO-B substrate phenylethylamine and the inhibitor N-(2-aminoethyl)-5-chloro-2-pyridine carboxamide . HCl (lazabemide) when compared to wild-type MAO-B. No major changes were observed in the kcat values of these chimeras. From the data obtained, two sequences, i.e. 62-103 and 146-220, appeared of particular importance in constituting the binding site of MAO-B. On the other hand, the catalytic properties and specificity of MAO-A appeared to be relatively insensitive to substitution of both the NH2- (up to position 112) and COOH-termini (from residue 395) of MAO-A with the corresponding MAO-B sequences. However, further modification of the central 283-residue sequence of MAO-A did not appear compatible with enzymic activity. None of the engineered chimeras showed a shift in specificity from one isoform to the other.
Gottowik
J
J
Pharma Division, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
Malherbe
P
P
Lang
G
G
Da Prada
M
M
Cesura
A M
AM
D001483:Base Sequence; D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D008995:Monoamine Oxidase / Q000737:chemistry* / Q000502:physiology; D008996:Monoamine Oxidase Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship
D008996:Monoamine Oxidase Inhibitors; D011993:Recombinant Fusion Proteins; D008995:Monoamine Oxidase