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bioRxiv · 10.1101/2023.03.13.532365

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

Stocker, C.·Khatanbaatar, T.·Würth-Roderer, K.·Cordara, G.·Krengel, U.·Kast, P.
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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 non-pathogenic {gamma}- or {beta}-proteobacteria. In {gamma}-proteobacterial fusion enzymes, the CM domain is N-terminal to the CDT domain, whereas in {beta}-proteobacteria the order is inversed. The CM domains share 15-20% sequence identity with the AroQ{gamma} 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 {micro}M, much lower than for *MtCM, whereas kinetic parameters are similar for CDT domains and PheC. There is no feedback inhibition of CM or CDT by the pathways end product Phe, and no catalytic benefit of the domain fusion compared to engineered single-domain constructs. The fusion enzymes of Aequoribacter fuscus, Janthinobacterium sp. HH01, and Duganella sacchari were crystallized and their structures refined to 1.6, 1.7, and 2.4 [A] resolution, respectively. Neither the crystal structures nor size-exclusion chromatography show evidence for substrate channeling or higher oligomeric structure that could account for cooperation of CM and CDT active sites. The genetic neighborhood with genes encoding transporter and substrate binding proteins suggests that these exported bifunctional fusion enzymes may participate in signaling systems rather than in the biosynthesis of Phe.

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Stocker, C., Khatanbaatar, T., Würth-Roderer, K., Cordara, G., Krengel, U., Kast, P.. 2023-03-13. Novel exported bifunctional fusion enzymes with chorismate mutase and cyclohexadienyl dehydratase activity: shikimate pathway enzymes teamed up in no man's land. https://doi.org/10.1101/2023.03.13.532365

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