Abstract
Chorismate synthase, the last enzyme in the shikimate pathway, catalyzes the transformation of 5-enolpyruvylshikimate 3-phosphate to chorismate, a biochemically unique reaction in that it requires reduced FMN as a cofactor. Here we report on the cloning, expression, and characterization of the protein for the first time from an extremophilic organism Thermotoga maritima which is also one of the oldest and most slowly evolving eubacteria. The protein is monofunctional in that it does not have an intrinsic ability to reduce the FMN cofactor and thereby reflecting the nature of the ancestral enzyme. Circular dichroism studies indicate that the melting temperature of the T. maritima protein is above 92 degrees C compared with 54 degrees C for the homologous Escherichia coli protein while analytical ultracentrifugation showed that both proteins have the same quaternary structure. Interestingly, UV-visible spectral studies revealed that the dissociation constants for both oxidized FMN and 5-enolpyruvylshikimate 3-phosphate decrease 46- and 10-fold, respectively, upon heat treatment of the T. maritima protein. The heat treatment also results in the trapping of the flavin cofactor in an apolar environment, a feature which is enhanced by the presence of the substrate 5-enolpyruvylshikimate 3-phosphate. Nevertheless, stopped-flow spectrophotometric 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 thermostability while it has conserved features relevant to its catalytic function.
Highlights
Chorismate synthase catalyzes the final step in the shikimate pathway, which links the metabolism of carbohydrates to the biosynthesis of the three aromatic amino acids and many aromatic secondary metabolites in a series of seven enzymatic steps
The expression of T. maritima chorismate synthase in BL21-CodonPlusTM(DE3)-RIL cells is ϳ6-fold greater than that observed in BL21(DE3) (Fig. 1, panel B, compare protein loaded in lanes 4 and 8)
The identity of the purified band as T. maritima chorismate synthase (41.7 kDa) was confirmed by N-terminal sequencing analysis (15 cycles of 50 pmol of protein resolved the amino acids MKLTIAGDSHGKYMV, which are identical to the data base entry for this chorismate synthase, accession number Q9WYI2), and the protein was estimated to be greater than 95% pure, as judged from SDS-PAGE (Fig. 2, lane 6)
Summary
Chorismate synthase catalyzes the final step in the shikimate pathway, which links the metabolism of carbohydrates to the biosynthesis of the three aromatic amino acids and many aromatic secondary metabolites in a series of seven enzymatic steps. It has been suggested that the ancestral enzyme harbored the intrinsic flavin reductase activity (i.e. was bifunctional) as it is hard to imagine how this activity could have evolved in a framework of what are known to be monofunctional enzymes, i.e. the bacterial and plant chorismate synthases [12]. Classification of T. maritima chorismate synthase with regard to how it acquires the reduced FMN cofactor can be considered to reflect the nature of the ancestral enzyme. We report the cloning, expression, and purification of chorismate synthase from the thermophilic eubacterium T. maritima. This is the first time that this enzyme has been described from an extremophilic organism. We report on the quaternary structure of T. maritima chorismate synthase
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