Elucidation of the trigonelline degradation pathway reveals previously undescribed enzymes and metabolites. N. Perchat et al, PNAS 2018 May 8;115(19).

Trigonelline (or N-methylnicotinate, or N-methylnicotinate-3-carboxylate) is a natural compound produced in particular by terrestrial plants and in the marine environment, where it plays an osmo-protective role. This molecule has attracted much attention in recent decades for its pharmacological potential, but microbiologists have known its role as a nutrient for much longer. Yet, curiously, no indication of its degradation pathway was available in metabolic databases or in the scientific literature. 

We used the laboratory's bacterial model, Acinetobacter baylyi ADP1, a soil bacterium with a sequenced genome and for which we have a complete bank of mutant strains by gene deletion, to identify a group of genes responsible for the use of trigonellin.

After producing the proteins in recombinant form, we were able to reconstitute the metabolic pathway in vitro and analyze it using an a priori-free metabolomic approach and then an extensive enzymatic study. Two totally unknown molecules, whose structure has been elucidated by NMR, have emerged as the first intermediates of the pathway, which ultimately leads to the formation of succinate and methylamine. Contrary to what might have been implied by the structural relationship of trigonellin with nicotinic acid, the degradation of trigonellin does not converge towards this compound but reveals the use by bacteria of another mode of cleavage of the pyridinic cycle.

In the end, while we had no prior indication of the course of the pathway, we clarified the order and nature of the reactions, described the intermediate metabolites of the pathway, highlighted the existence of this pathway in bacterial species encountering trigonellin in their environment and revealed a new panoply of metabolic creativity shown by the microbial world. 

Figure legend: TG degradation pathway in ADP1. TgnA and TgnB, two-component TG oxygenase; TgnC, MFMB dehydrogenase; TgnD, MFMS hydrolase; TgnE, succinate semialdehyde dehydrogenase; TgnF, succinate semialdehyde dehydrogenase stimulating protein. Data from NMR analysis for MFMB and MFMS are indicated in grey boxes. A and A’ represent the two closed forms conformers of MFMB detected by NMR. B and B’ represent the two conformers of MFMS. TgnF is indicated in brackets since it is not known whether it is an enzyme.