title: Functional expression of C-terminally truncated human monoamine oxidase type A in Saccharomyces cerevisiae.
abstract: The deduced amino acid sequence of human liver monoamine oxidase type A was analyzed with secondary structure programs. These analyses and comparison to other flavoproteins identified a single potential transmembrane hydrophobic peptide at the C-terminus suggesting that this peptide is a membrane anchor and that the remainder of the protein constitutes a soluble domain. Truncation of the C-terminus by 24 amino acids which are inclusive of the putative transmembrane peptide, however, gave a protein which exhibited solubility properties substantially similar to the wild type enzyme. This result indicates that the hydrophobic behavior of monoamine oxidase type A is due to more complex features than a single transmembrane anchor. The mutant enzyme expressed in yeast appears to form a disulfide bond which reduces catalytic efficiency by up to 90%. Full activity, however, can be recovered by incubation with dithiothreitol, suggesting that in the wild type enzyme the amino acid residues deleted in the mutant protein protect two cysteine residues (those involved in the formation of the disulfide bond in the mutant) from oxidation and that the deleted residues are in close proximity to the active site. The activation experiments indicated that the deleted amino acids do not contribute any catalytic residues to the active site.
authors.affiliation: Molecular Biology Division, Veterans Affairs Medical Center, San Francisco, California.
authors.forename: W
authors.initials: W
authors.lastname: Weyler
mesh_terms: D055598:Chemical Phenomena; D002621:Chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation; D017353:Gene Deletion; D008995:Monoamine Oxidase / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D008995:Monoamine Oxidase; D004229:Dithiothreitol