Chorismate synthase from the hyperthermophile Thermotoga maritima combines thermostability and increased rigidity with catalytic and spectral properties similar to mesophilic counterparts.
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.
ETH-Zürich, Institut für Pflanzenwissenschaften, Universitätstrasse 2, CH-8092 Zürich, Switzerland.
T B
TB
Fitzpatrick
P
P
Killer
R M
RM
Thomas
I
I
Jelesarov
N
N
Amrhein
P
P
Macheroux
D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism; D002384:Catalysis; D004795:Enzyme Stability; D019761:Phosphorus-Oxygen Lyases / Q000032:analysis / Q000378:metabolism*; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology*
D001426:Bacterial Proteins; C019895:chorismate synthase; D019761:Phosphorus-Oxygen Lyases