Substrate channelling in catabolism of methylated amines

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.

The oxidation of methylated-amines can lead to the formation of toxic formaldehyde through the hydrolysis of unstable iminium products. Several organisms appear to avoid formaldehyde production by using bifunctional amine oxidases that contain a 5,10-methylene THF synthase domain fused to the amine oxidase domain. In this case, the oxidized C1-unit is transferred to tetrahydrofolate rather than being released as formaldehyde following iminium hydrolysis. We recently determined the first crystal structure of a member of this enzyme family (DMGO) and have shown it to contain a simple architecture that could support a channelling mechanism that avoids mass leakage of the oxidized methylated amine into the bulk solvent. We will invetsigate both in vitro and in vivo if, and to what extent, substrate channelling occurs in these enzymes. We plan to characterize additional members of this enzyme family (e.g. dimethylglycine dehydrogenase, DMGDH and N-methylaminobutyrate oxidase, MABO) for which (i)we have obtained diffraction quality crystals and (ii) shown markedly distinct behaviour in solution when compared to DMGO (e.g. no inhibition of amine oxidase activity by reduced folates). Using kinetic crystallography we aim to determine the mechanism of 5,10-methylene THF synthesis in these enzymes. Furthermore, we aim to provide conclusive insight into the substrate channelling in these enzymes by analysing the in vivo properties of selected mutant forms of structural elements essential to this mechanism. In addition, structural and mechanistic insights will be sought in structurally distinct members of this family, e.g. the pyruvate dehydrogenase phosphate complex and a fusion between a conserved protein of unknow function and the 5,10-methylene THF synthase domain. The results from this research should provide a detailed understanding of the mechanism and evolution of substrate channeling and guide future protein engineering desig