aroG, encoding the monofunctional chorismate mutase (TtCM) of the thermophilic gram-negative bacterium Thermus thermophilus, was cloned and its gene product characterized. TtCM was purified to homogeneity on an SDS polyacrylamide gel as a His-fusion protein with a deduced molecular mass of 15.8 kDa. The enzyme belongs to the rare group of AroH-type chorismate mutases which are mainly found in gram-positive bacteria of the Bacillus/Clostridia group and have recently also been described for gram-negative organisms. The native molecular mass is consistent with a pseudo-α/β barrel enzyme that is organized as a trimer. Comparison of the enzyme’s structure with that of its mesophilic counterpart from Bacillus revealed an increase in hydrophilicity on the protein’s surface, greater hydrophobicity in cavities within the protein, and greater restriction of conformational freedom, features that contribute to the thermal stability of this chorismate mutase. The kinetic data show Michaelis-Menten substrate saturation with a K m of 290 μM, and a k cat/K m value of 180 s−1 mM−1. TtCM was inhibited by tyrosine with a K i =34 μM, possibly in a competitive manner.
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We thank Prof. Wolfgang Liebl for providing the wild-type T. thermophilus strain, Andrea Pfeil for assistance in enzyme assays and the other members of the laboratory, especially Dr. Sven Krappmann, for helpful discussions. This work was supported by grants from the Deutsche Forschungsgemeinschaft, the Volkswagen-Stiftung, the niedersächsische Vorab der Volkswagen-Stiftung, and the Fonds der Chemischen Industrie.
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Helmstaedt, K., Heinrich, G., Merkl, R. et al. Chorismate mutase of Thermus thermophilus is a monofunctional AroH class enzyme inhibited by tyrosine. Arch Microbiol 181, 195–203 (2004). https://doi.org/10.1007/s00203-003-0639-z