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

Christian Stocker; Tamjidmaa Khatanbaatar; Luca Bressan; Kathrin Würth-Roderer; Gabriele Cordara; Ute Krengel; Peter Kast

doi:10.1016/j.jbc.2023.105161

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

J Biol Chem. 2023 Oct;299(10):105161. doi: 10.1016/j.jbc.2023.105161. Epub 2023 Aug 14.

Authors

Christian Stocker¹, Tamjidmaa Khatanbaatar², Luca Bressan¹, Kathrin Würth-Roderer¹, Gabriele Cordara², Ute Krengel³, Peter Kast⁴

Affiliations

¹ Laboratory of Organic Chemistry, D-CHAB, ETH Zurich, Zurich, Switzerland.
² Department of Chemistry, University of Oslo, Oslo, Norway.
³ Department of Chemistry, University of Oslo, Oslo, Norway. Electronic address: ute.krengel@kjemi.uio.no.
⁴ Laboratory of Organic Chemistry, D-CHAB, ETH Zurich, Zurich, Switzerland. Electronic address: kast@org.chem.ethz.ch.

Abstract

Chorismate mutase (CM) and cyclohexadienyl dehydratase (CDT) catalyze two subsequent reactions in the intracellular biosynthesis of l-phenylalanine (Phe). Here, we report the discovery of novel and extremely rare bifunctional fusion enzymes, consisting of fused CM and CDT domains, which are exported from the cytoplasm. Such enzymes were found in only nine bacterial species belonging to non-pathogenic γ- or β-Proteobacteria. In γ-proteobacterial fusion enzymes, the CM domain is N-terminal to the CDT domain, whereas the order is inverted in β-Proteobacteria. The CM domains share 15% to 20% sequence identity with the AroQ_γ class CM holotype of Mycobacterium tuberculosis (∗MtCM), and the CDT domains 40% to 60% identity with the exported monofunctional enzyme of Pseudomonas aeruginosa (PheC). In vitro kinetics revealed a K_m <7 μM, 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 pathway's end product Phe, and no catalytic benefit of the domain fusion compared with 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 Å resolution, respectively. Neither the crystal structures nor the size-exclusion chromatography show evidence for substrate channeling or higher oligomeric structure that could account for the 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.

Keywords: X-ray crystallography; bacterial metabolism; cooperative active site; enzyme kinetics; exported chorismate mutase; fusion protein; multifunctional enzyme; prephenate dehydratase; shikimate pathway; substrate channeling.