Insights into Monoamine Oxidases: Biocatalytic Potential and Evolutionary Diversification

Monoamine oxidases (MAOs) are flavin-dependent enzymes that catalyze oxidative deamination of primary and secondary amines to the corresponding aldehydes or ketones with the release of ammonia and hydrogen peroxide. In mammals, MAOs are part of the outer mitochondrial membrane and occur in two isoforms, MAO A and MAO B, which are pivotal in neurotransmitter metabolism and are directly implicated in neurological events and age-related disorders. Besides their biological significance, MAOs and their homologues have also seen growing interest as multi- functional biocatalysts. Their capacity for selective amine oxidations has found applications in the preparation of enantiopure drug intermediates, in biosensors, and in green chemistry. The first part of this thesis (Chapter 1) introduces the structural and biochemical features of MAOs within the frame of the broader family of FAD-dependent amine oxidases. It points out the distribution and the physiological function of mammalian isoforms, analyzes homologous enzymes in fungi and bacteria, and considers their possible applications in biocatalysis and biosensing. This introduction prepares the reader for the subsequent experimental chapters, which deal with both the applied and evolutionary sides of the MAO family. One of the primary focuses of this thesis has been the characterization of a novel thermostable bacterial enzyme, MAOTb, in a thermophilic Thermoanaerobacterales isolate (Chapter 3). MAOTb is distinguished by high expression levels in Escherichia coli and high thermostability, with retention of activity at temperatures greater than 70 °C. Biochemical characterization revealed an unequivocal preference for long- chain aliphatic amines, n-heptylamine being its most active substrate. Pre-steady- state kinetics experiments verified that the reduced enzyme reacts rapidly with molecular oxygen, making it a bona fide oxidase. Structural comparison at 1.5 Å resolution revealed close similarity with human MAOs, e.g., conserved aromatic cage and covalent FAD ligation, but also distinguishing features such as absence of membrane-anchoring helix, which accounts for its solubility. Such features make MAOTb a potential candidate for industrial and synthetic applications. To investigate its engineering potential, structure-based mutagenesis was used (Chapter 4). Targeting critical residues that determine the active site from the substrate-bound crystallographic structure, variants with altered activity against aromatic monoamines were realized, demonstrating the feasibility of substrate specificity adjustment in this enzyme. These discoveries emphasize the suitability of MAOTb as a robust scaffold for tailoring oxidase activity toward drugs of pharmaceutical value. Concurrently with this, the thesis also explores the evolutionary diversification of the MAO family (Chapter 5). Phylogenetic analysis revealed that the duplication event resulting in MAO A and MAO B occurred in the tetrapod ancestor, while non- tetrapod jawed vertebrates retained the ancestral gene (MAO A/B) along with a new paralog, MAO F. Functional investigation of recombinant MAO A/B and MAO F in representative species revealed that both have human MAO A-like features but with distinct enzymatic properties. These findings contribute to our understanding of vertebrate MAO evolutionary history and uncover a novel lineage of putative physiological importance. In general, this thesis presents new insights into the monoamine oxidase family from complementary perspectives. The discovery and engineering of MAOTb introduce an intriguing new biocatalyst with industrial application, while the evolutionary analysis leading to the identification of MAO F further illuminates vertebrate molecular evolution. By combining different approaches, this work identifies a path spanning basic and applied research and illustrates both the ancient diversification and the modern utility of monoamine oxidases.