Novel exported bifunctional fusion enzymes with chorismate mutase and cyclohexadienyl dehydratase activity: shikimate pathway enzymes teamed up in no man’s land

Abstract

Chorismate mutase (CM) and cyclohexadienyl dehydratase (CDT) catalyze two subsequent reactions in the intracellular biosynthesis of phenylalanine. Surprisingly, exported CMs and CDTs exist in bacterial pathogens. Here, we report the discovery of novel and extremely rare exported bifunctional fusion enzymes, consisting of fused CM and CDT domains. Such enzymes were found in only nine bacterial species belonging to nonpathogenic γ- or β-proteobacteria. In γ-proteobacterial fusion enzymes, the CM domain is Nterminal to the CDT domain, whereas in β-proteobacteria the order is inversed. The CM domains share 15-20% sequence identity with the AroQγ class CM holotype of Mycobacterium tuberculosis (*MtCM), and the CDT domains 40-60% identity with the exported monofunctional enzyme of Pseudomonas aeruginosa (PheC). In vitro kinetics revealed a Km <7 μM, much lower than for *MtCM, whereas kinetic parameters are similar for CDT domains and PheC