Isolation and characterization of an evolutionary precursor of human MAO A and B

Singer T.P., Yankovskaya V.L., Bernard S.S., Cronin C.C., Sablin S.O.
PubMed Id: 9503562
Year: 1997  Volume: 43  Issue: 6  Pages: 440-456
An interesting flavoprotein-type monoamine oxidase (MAO) was recently isolated from Aspergillus niger and cloned by Schilling and Lerch (1995a,b) The properties of this MAO, as well as a substantial part of its amino acid sequence resemble those of both MAO A and B from higher animals, raising the possibility that it may be an evolutionary precursor of these mitochondrial enzymes. It differs from MAO A and B in several respect, however, including the fact that it is soluble and of peroxisomal localization and that the FAD is non-covalently attached. We have overexpressed the fungal enzyme (MAO-N) in Escherichia coli, isolated it for the first time in pure form, and, in collaboration with Dr. Elena Sablin, crystallized it. Since several of the observations of previous workers on MAO-N could not be reproduced and seem to be erroneous, we have reexamined its, substrate specificity, interaction with reversible and irreversible inhibitors and other catalytic and molecular 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, while others are similar to MAO A, including biphasic kinetics in double reciprocal plots. Contrary to the report of Schilling and Lerch (1995a), however, the fungal enzyme does not oxidize serotonin, norepinephrine, dopamine or other biogenic amines. MAO-N is irreversibly inhibited by stoichiometric amounts of both (-)deprenyl and clorgyline in a mechanism-based reaction, forming flavocyanine adducts with N(5) of the FAD, like the mammalian enzymes, but inactivation is much faster with clorgyline than deprenyl, suggesting again a closer resemblance to MAO A than B. The dissociation constants for a large number of reversible competitive inhibitors have been determined for MAO-N and comparison with similar values for MAO A and B again pointed to a much greater similarity to the former than the latter. Experiments designed to change the linkage of the FAD to covalent form by site-directed mutagenesis and to dissociate.
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Keywords: monoamine oxidase N, purification, expression, molecular properties, site-directed mutagenesis

Singer, T. P., Yankovskaya, V. L., Bernard, S. S., Cronin, C. C., Sablin, S. O. (1997). Isolation and characterization of an evolutionary precursor of human MAO A and B. Voprosy Meditsinskoi Khimii, 43(6), 440-456.
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