CLONING, SEQUENCING AND HETEROLOGOUS EXPRESSION OF THE MONOAMINE-OXIDASE GENE FROM ASPERGILLUS-NIGER

The gene encoding the flavin-containing monoamine oxidase (MAO-N) of t he filamentous fungus Aspergillus niger was cloned. MAO-N is the first nonvertebrate monoamine oxidase described to date. Three partial cDNA clones, isolated from an expression library, were used to identify an d clone the structural gene (maoN) from an A. niger genomic DNA librar y. The maoN gene was sequenced, and analysis revealed an open reading frame that codes for a protein of 495 amino acids with a calculated mo lecular mass of 55.6 kDa. Sequencing of an internal proteolytic fragme nt of the purified enzyme confirmed the derived amino acid sequence. A nalysis of the deduced amino acid sequence indicates that MAO-N is str ucturally related to the human monoamine oxidases MAO-A and MAO-B. In particular, the regions known to be involved in the binding of the FAD cofactor show a high degree of homology; however, the conserved cyste ine residue to which the flavin cofactor is covalently bound in the ma mmalian forms is absent in the fungal enzyme. MAO-N has the C-terminal tripeptide Ala-Arg-Leu, which corresponds to the consensus targeting sequence found in many peroxisomal enzymes. The full-length cDNA for M AO-N was expressed in Escherichia coli from the T7 promoter of the exp ression vector pET3a, yielding a soluble and fully active enzyme form.