A Small, Thermostable, and Monofunctional Chorismate Mutase from the Archeon Methanococcus jannaschii

Abstract

The gene for chorismate mutase (CM) from the archeon Methanococcus jannaschii, an extreme thermophile, was subcloned and expressed in Escherichia coli. This gene, which belongs to the aroQ class of CMs, encodes a monofunctional enzyme (AroQ_f) able to complement the CM deficiency of an E. coli mutant strain. The purified protein follows Michaelis−Menten kinetics (k_cat = 5.7 s^-1 and K_m = 41 μM at 30 °C) and displays pH-independent activity in the range of pH 5−9. Its activation parameters [ΔH^⧧ = 16.2 kcal/mol, ΔS^⧧ = −1.7 cal/(mol·K)] are similar to those of another well characterized AroQ class CM, the mesophilic AroQ_p domain from E. coli. Like AroQ_p, the thermophilic CM is an α-helical dimer, but approximately 5 kcal/mol more stable than its mesophilic counterpart as judged from equilibrium denaturation studies. The possible origins of the thermostability of M. jannaschii AroQ_f, the smallest natural CM characterized to date, are discussed in light of available sequence and tertiary structural information.