ISOLATION AND CHARACTERIZATION OF AN EVOLUTIONARY PRECURSOR OF HUMAN MONOAMINE OXIDASE-A AND OXIDASE-B

An interesting flavoprotein-type monoamine oxidase (MAO) was recently isolated from Aspergillus niger and cloned [Schilling, B. & Lerch, K. (1995a) Biochim. Biophys, Acta 1243, 529-537; Schilling, B. & Lerch, K . (1995b) Mol. Gen. Genet. 247, 430-438]. The properties of this MAO, as well as a substantial part of its amino acid sequence, resemble tho se of both MAO A and B from higher animals, raising the possibility th at it may be an evolutionary precursor of these mitochondrial enzymes. It differs from MAO A and B in several respects, however, including t he fact that it is soluble and of peroxisomal location and that the FA D is non-covalently attached. We have overexpressed the fungal enzyme (MAO-N) in Escherichia coli and isolated it in pure form. Since severa l of the observations of previous workers On MAO-N could not be reprod uced, we have reexamined its substrate specificity, interaction with r eversible and irreversible inhibitors and ether catalytic and molecula r properties. MAO-N has a considerably higher turnover number on many aliphatic and aromatic amines than either form of the mammalian enzyme . Some aspects of the substrate specificity resemble those of MAO B, w hile others are similar to MAO A, including biphasic kinetics in doubl e reciprocal plots. Contrary to a previous report [Schilling, B. & Ler ch. K. (1995a) Biochim, Biophys. Acta 1243, 529-537], however, the fun gal enzyme does not oxidize serotonin, nonpinephrine, dopamine or othe r biogenic amines. MAO-N is irreversibly inhibited by stoichiometric a mounts of both (-)deprenyl and clorgyline in a mechanism-based reactio n, forming flavocyanine adducts with N5 of the FAD, like the mammalian enzymes, but inactivation is much faster with clorgyline than depreny l, suggesting a closer resemblance to MAO A than B. The dissociation c onstants for a large number of reversible competitive inhibitors have been determined for MAO-N and comparison with similar values far MAO A and B again pointed to a greater similarity to the farmer than the la tter.