Pseudomonadal itaconate degradation gene cluster encodes enzymes for methylsuccinate utilization

Branched-chain C₅-dicarboxylic acids (e.g., citramalate, mesaconate or methylsuccinate) and their CoA-esters are important intermediates in bacterial metabolism, while itaconate is an antimicrobial agent, a potent immunomodulator and a growth substrate for many bacteria. The itaconate degradation pathway consists of three reactions catalyzed by itaconate CoA transferase, itaconyl-CoA hydratase and (S)-citramalyl-CoA lyase encoded in a cluster, which in saprophytic bacteria contains two additional genes for a putative acyl-CoA dehydrogenase and a protein of the MmgE/PrpD family. Here, we heterologously produced the corresponding proteins from Cupriavidus necator and Pseudomonas aeruginosa and showed that they catalyze the (RS)-methylsuccinyl-C4-CoA dehydrogenase and an (S)-(R)-methylsuccinate isomerase reaction, respectively. Together with itaconate CoA transferase, which is highly active with (R)-methylsuccinate but has low activity with (S)-methylsuccinate, these enzymes allow the utilization of both stereoisomers of methylsuccinate. Our bioinformatic analysis revealed that 1.6% of the sequenced prokaryotes (mainly Betaproteobacteria) possess an identified methylsuccinate isomerase. Analysis of the conserved amino acids of methylsuccinate isomerase and other MmgE/PrpD proteins suggests that they share a common catalytic mechanism via the formation of an enolate intermediate. The presence of specific methylsuccinate utilization genes in the itaconate degradation cluster, which is widespread in saprophytic bacteria, suggests the importance of methylsuccinate in the environment.