Structure/Function Relationships of Mitochondrial Monoamine Oxidase A and B Chimeric Forms

Abstract

Monoamine oxidases (MAO) A and B show a high degree of amino acid similarity. Apart from the NH,-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 NH,-terminus of one M A 0 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 M A 0 isoforms, chimeras with increasing length of the NH,-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 . HC1 (lazabemide) when compared to wild-type MAO-B. No major changes were observed in the k,,, 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 NH,-(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.

Molecular and Mechanistic Properties of the Membrane-Bound Mitochondrial Monoamine Oxidases

“…These structural data support previous biochemical data (29) that identified the C-terminal helix as the mode of MAO A or MAO B binding to the outer mitochondrial membrane. It is of interest that chimeric enzymes constructed using fragments of MAO A and MAO B show that “swaps” of the respective C-terminal helices between MAO B to MAO A result in inactive enzyme (30, 31) suggesting that there are differences in the specific membrane-binding architectures between the two isozymes.…”