Itaconate, a product of mammalian macrophages, controls immune responses and serves as an antimicrobial agent by inhibiting various reactions of carbon metabolism [1]. Some pathogens, including Pseudomonas aeruginosa, metabolize itaconate, detoxifying it and using it as a growth substrate [2]. Interestingly, the P. aeruginosa itaconate utilization operon contains three genes of unknown function in addition to the genes encoding the itaconate degradation enzymes, itaconate CoA-transferase, itaconyl-CoA hydratase and citramalyl-CoA lyase. Two of the unknown genes, encoding putative acyl-CoA dehydrogenase and MmgE protein, are found in a homologous gene cluster in Cupriavidus necator H16 (E6A55_22600 and E6A55_22590, respectively, referred further as mcd and msi). It has been proposed that the P. aeruginosa dehydrogenase catalyzes methylsuccinyl‑CoA oxidation [2], whereas the corresponding MmgE protein may function as a methylsuccinate or methylsuccinyl-CoA isomerase [3]. Here, we showed that mcd from C. necator encodes a methylsuccinyl-C4-CoA dehydrogenase that catalyzes the conversion of both (S)- and (R)‑enantiomers to mesaconyl-C4-CoA (k_cat/K_M of 1.2 and 1.4 s^-1 mM^-1 for (S)- and (R)-methylsuccinyl-CoA, respectively). In contrast, the C. necator itaconate CoA-transferase worked preferentially with (R)‑methylsuccinate (k_cat/K_M of 0.7 and 64.1 s^-1 mM^-1 for (S)- and (R)‑isomers, respectively). In its turn, the C. necator MmgE protein interconverted (S)‑ and (R)‑methylsuccinate, but not methylsuccinyl-CoA, thus allowing the efficient conversion of (S)‑methylsuccinate through the pathway. Indeed, both stereoisomers of methylsuccinate could be used by C. necator as the sole carbon source. We confirmed that the P. aeruiginosa homologs had the same function, thus revealing methylsuccinate degradation as the second function of the itaconate utilization operon in pseudomonads. Since this operon is widespread among bacteria, methylsuccinate appears to be an important natural metabolite, the source of which remains to be determined.

[1] Cordes et al, Annu Rev Nutr 35:451 (2015).

[2] Sasikaran et al, Nat Chem Biol 10:371 (2014).

[3] de Witt et al, Metab Eng 75:205 (2023).