The vast number of poorly characterised enzymes in Mycobacterium tuberculosis (Mtb) is one of the key barriers precluding a better understanding of the biology that underpins pathogenesis. Here, we investigated the Mtb orphan enzyme Rv2498c to delineate its physiological role. Our results from in vitro enzymatic assays, phylogenetic analysis, X-ray crystallography and in vivo Mtb experiments, de-orphan Rv2498c as a multi-functional β-hydroxyacyl-CoA lyase/thioesterase (β-HAClyase/thioesterase) that participates in three different metabolic pathways: L-leucine catabolism, itaconate dissimilation, and glyoxylate shunt. Moreover, the deletion of the rv2498c gene from the Mtb genome resulted in attenuation in the mouse model compared to infection with the parent strain. To the best of our knowledge, this is the first report of an (R)-3-hydroxyl-3-methylglutaryl-CoA for leucine catabolism and an itaconate-specific resistance mechanism in Mtb.