Substrate channelling in catabolism of methylated amines

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The many chemical reactions supporting life are catalysed by enzymes. Some of the reactions that occur naturally can give rise to unstable compounds. If left unattented by the cellular machinery, this can be toxic to the host organism. To avoid this, many enzymes guide these unstable compounds to the next enzyme in a process called substrate channelling. This involves the careful and intricate design of enzymes at the molecular level where the unstable compound is transferred through narrow channels to its final destination. These channels ensure the compound does not escape into the bulk solution and thus does not undergo unwanted side reactions. How these enzyme systems have evolved is uncertain, although it has been proposed that the first 'channelling enzymes' were far from perfect, leaking unstable compounds at acceptable levels. We have recently elucidated the molecular detail of an enzyme (DMGO) that apparently mimics one of these early designs of a substrate channelling enzyme. IThe structure of DMGO is far simpler than other known channelling systems, yet it is widespread among organisms including humans, suggesting further significant improvement does not provide additional benefit to the organism or has not been possible to achieve. The protein in question catalyses the oxidation of methylated-amines. Enzymic oxidation of the methyl group can, over time, give rise to the release of toxic formaldehyde. To avoid this, the enzyme channels the oxidized product to a second active site where it transfers the oxidized methyl-group to tetrahydrofolate, an essential co-enzyme that acts as a resoervoir of one-carbon units. We will investigate if these simple enzymes leak unstable intermediate compounds as their simple design would suggest. Furthermore, we aim to gain atomic level insight into function of the molecular architecture by determining the structure of other members of this protein family. Using sophisticated structural biology techniques we aim to visualise what happens at active site 2 (the folate site) during transfer of oxidized-methyl groups. We will also focus our efforts on characterizing other proteins containing some key elements present in the bifunctional amine oxidases. We thus wish to determine whether this particular simple substrate channelling mechanism is widespread and has evolved in several distinct systems. Information gained from our studies will provide insight into the origins and mechanisms of substrate channelling, and provide a detailed understanding of channelling that could guide the next level of protein design and engineering through the incorporation of retaining nano-sized reservoirs in protein catalysts.