Choline degradation inParacoccus denitrificans: identification of sources of formaldehyde

Abstract

ABSTRACTParacoccus denitrificansis a facultative methylotroph that can grow on methanol and methylamine as sole sources of carbon and energy. Both are oxidized to formaldehyde and then to formate, so growth on C1 substrates induces the expression of genes encoding enzymes required for the oxidation of formaldehyde and formate. Catabolism of some complex organic substrates (for example choline and L-proline betaine) generates formaldehyde, which is oxidized to CO2, likely by the same enzymes that oxidize formaldehyde generated from C1 substrates. Thus, regulatory mutants that fail to induce expression of the formaldehyde catabolic enzymes cannot grow on methanol, methylamine and choline. The pathway for the oxidation of choline to glycine includes three steps that may be oxidative demethylations that generate formaldehyde. However, among characterized choline degradation pathways in several organisms there are variants of these reactions that do not generate formaldehyde. Therefore, the number of equivalents and the sources of formaldehyde made during choline degradation inP. denitrificansare not known. By exploring mutant phenotypes and the activities of a promoter and enzyme known to be up-regulated by formaldehyde, we identify the oxidations of glycine betaine and dimethylglycine as sources of formaldehyde. Genetic evidence implicates two orthologous monooxygenases in the oxidation of glycine betaine. Interestingly, one of these appears to be a bifunctional enzyme that also oxidizes L-proline betaine (stachydrine). We present preliminary evidence to suggest that growth on L-proline betaine induces expression of a formaldehyde dehydrogenase distinct from the enzyme induced during growth on other formaldehyde-generating substrates.IMPORTANCEThe bacterial degradation of one carbon compounds (methanol and methylamine) and of some complex multi-carbon compounds (for example, choline) generates formaldehyde. Formaldehyde is toxic and must be removed, which can be done by oxidation to formate and then to carbon dioxide. These oxidations provide a source of energy, in some species the CO2thus generated can be assimilated into biomass. Using the Gram-negative bacteriumParacoccus denitrificansas the experimental model, we show that oxidation of choline to glycine generates two equivalents of formaldehyde and we identify the two steps in the catabolic pathway that are responsible. Our work sheds further light on metabolic pathways that are likely important in a variety of environmental contexts.