Chorismate synthase from the hyperthermophile Thermotoga maritima combinesthermostability and increased rigidity with catalytic and spectral properties similar to mesophilic counterparts

Chorismate synthase, the last enzyme in the shikimate pathway, catalyzes th e transformation of 5-enolpyruvylshikimate 3-phosphate to chorismate, a bio chemically unique reaction in that it requires reduced FMN as a cofactor. H ere we report on the cloning, expression, and characterization of the prote in for the first time from an extremophilic organism Thermotoga maritima wh ich is also one of the oldest and most slowly evolving eubacteria, The prot ein is monofunctional in that it does not have an intrinsic ability to redu ce the FMN cofactor and thereby reflecting the nature of the ancestral enzy me. Circular dichroism studies indicate that the melting temperature of the T. maritima protein is above 92 degreesC compared with 54 degreesC for the homologous Escherichia coli protein while analytical ultracentrifugation s howed that both proteins have the same quaternary structure, Interestingly, UV-visible spectral studies revealed that the dissociation constants for b oth oxidized FMN and 5-enolpyruvylshikimate 3-phosphate decrease 46- and 10 -fold, respectively, upon heat treatment of the T, maritima protein. The he at treatment also results in the trapping of the flavin cofactor in an apol ar environment, a feature which is enhanced by the presence of the substrat e 5-enolpyruvylshikimate 3-phosphate, Nevertheless, stopped-flow spectropho tometric evidence suggests that the mechanism of the T. maritima protein is similar to that of the E. coli protein. In essence, the study shows that T . maritima chorismate synthase exhibits considerably higher rigidity and th ermostability while it has conserved features relevant to its catalytic fun ction.