An Internal Reaction Chamber in Dimethylglycine Oxidase Provides Efficient Protection from Exposure to Toxic Formaldehyde

Abstract

We report a synthetic biology approach to demonstrate substrate channeling in an unusual bifunctional flavoprotein dimethylglycine oxidase. The catabolism of dimethylglycine through methyl group oxidation can potentially liberate toxic formaldehyde, a problem common to many amine oxidases and dehydrogenases. Using a novel synthetic in vivo reporter system for cellular formaldehyde, we found that the oxidation of dimethylglycine is coupled to the synthesis of 5,10-methylenetetrahydrofolate through an unusual substrate channeling mechanism. We also showed that uncoupling of the active sites could be achieved by mutagenesis or deletion of the 5,10-methylenetetrahydrofolate synthase site and that this leads to accumulation of intracellular formaldehyde. Channeling occurs by nonbiased diffusion of the labile intermediate through a large solvent cavity connecting both active sites. This central "reaction chamber" is created by a modular protein architecture that appears primitive when compared with the sophisticated design of other paradigm substrate-channeling enzymes. The evolutionary origins of the latter were likely similar to dimethylglycine oxidase. This work demonstrates the utility of synthetic biology approaches to the study of enzyme mechanisms in vivo and points to novel channeling mechanisms that protect the cell milieu from potentially toxic reaction products.

Crystal structure of the bifunctional proline utilization A flavoenzyme from Bradyrhizobium japonicum

“…In particular, the irregular shape of the cavity is rather different from the tunnels that channel indole and ammonia in tryptophan synthase and carbamoyl phosphate synthetase (27,28). The PutA cavity better resembles that of dimethylglycine oxidase (29,30).…”

Glutathione metabolism links FOXRED1 to NADH:ubiquinone oxidoreductase (complex I) deficiency: A hypothesis

“…When DMGO is expressed in E. coli, very little formaldehyde is released. However, when its C-terminus is deleted, or when folate is absent, high levels of formaldehyde are generated (Tralau et al, 2009).…”

Formaldehyde degradation in Corynebacterium glutamicum involves acetaldehyde dehydrogenase and mycothiol-dependent formaldehyde dehydrogenase

“…Intracellularly generated formaldehyde occurs as a by-product of enzymic methylation and demethylation processes or due to the oxidation of methanol (Szende & Tyihák, 2010). Besides catabolism of Formaldehyde degradation in C. glutamicum methylated amino acids like S-adenosyl-L-methionine (Tyihák et al, 1998), E-N-trimethyl lysine and dimethylglycine (Tralau et al, 2009) and biosynthesis of glycine from serine via serine hydroxymethyltransferase (PetersWendisch et al, 2005) also aromatic compounds such as vanillate, which are natural carbon sources of C. glutamicum (Merkens et al, 2005), give rise to formaldehyde. Formaldehyde dissimilation was shown here to be essential for the vanillate metabolism in C. glutamicum such as is known for other organisms like P. putida (Hibi et al, 2005).…”