Chorismate mutase/prephenate

In E. coli and many other bacteria a second bifunctional enzyme, chorismate mutase-prephenate dehydrogenase causes the isomerization of chorismate and the oxidative decarboxylation of prephenate to p-hydroxyphenylpyruvate (steps h and /c, Fig. 25-l).39 The latter can be converted by transamination to tyrosine.40-42... 

The bacterial enzyme chorismate mutase-prephenate dehydrogenase is peculiar because it is a single protein unit with two catalytic activities. It catalyzes the sequential reactions of mutation of chorismate to prephenate and then the reaction that leads to the formation of phenylalanine and tyrosine, through oxidation of prephenate. The first of these reactions is interesting because it is one of the few strictly single-substrate enzymatic reactions it entails... 

Over the past decade, several strains of yeast [43, 44] and E. coli [45, 46] have been engineered that lack chorismate mutase. A typical bacterial selection system is depicted schematically in Fig. 3.5. It is based on E. coli strain KA12 [45], which has deletions of the chromosomal genes for both bifunctional chorismate mutases (chorismate mutase-prephenate dehydrogenase and chorismate mutase-prephenate dehydratase). Monofunctional versions of prephenate dehydratase [47] and prephenate dehydrogenase [48] from other organisms are supplied by the plasmid pKIMP-UAUC, leaving the cells deficient only in chorismate mutase activity [45]. 

Fig. 3.5. An engineered E. coli selection system lacking endogenous chorismate mutase activity [45]. The genes encoding the bifunctional enzymes chorismate mutase-prephenate dehydrogenase and chorismate mutase-prephenate dehydratase were deleted, and monofunctional versions of the dehydrogenase and dehydratase were supplied on plasmid pKIMP-UAUC. Poten-...

The aroF gene lies in an operon with tyrA, which encodes the bi functional protein chorismate mutase/prephenate dehydrogenase. Both genes arc regulated by the TyrR repressor protein complexed with tyrosine. The aroF gene product accounts for 80% of the total DAHP synthase activity in wild-type F. coli cells. 

It has been shown that both the catalyzed and the uncatalyzed reaction proceed through a chairlike transition state, stabilized in polar media59 60-143 262-263. Compound 1, an analog of the transition-state structure, proved to be a potent inhibitor of E. coli chorismate mutase-prephenate dehydrogenase204-255. For a discussion of the mechanism and structural requirements of the enzyme see refs 266 and 267. 

Enzymatic studies with chorismate mutase prephenate dehydrogenase from Escherichia coli show that the chorismate prephenate analog 7 is not a substrate for chorismate mutase65. Both 7 and 8 are moderately competitive inhibitors for chorismate mutase. Ester derivatives 4 and 5, as well as 6, readily undergo Claisen rearrangements in organic solvents. 

Vitamin Soybean [15] Overexpression of enzymes (chorismate mutase-prephenate dehydrogenase, homogentisate phytyltrans-ferase, and p-hydroxyphenylpyruvate dioxygenase) that are involved with the biosynthesis of vitamin E precursors (increase in tocodiromanols, including tocotrienol)... 

Chavez-Bejar, M.l. et al (2008) Metabolic engineering of Escherichia coli for L-tyrosine production by expression of genes coding for the chorismate mutase domain of the native chorismate mutase-prephenate dehydratase and a cyclohexadienyl dehydrogenase from Zymomonas... 

Baldwin, G. S. and B. E, Davidson, Kinetic studies on the mechanism of chorismated mutase/prephenate dehydratase from Escherichia coli, Biochim. Biophys. Acta, 742, 374-383 (1983). 

This realization has not escaped the bioorganic community, and a number of research groups, notably those of Andrews and Berchtold, have synthesized molecules that are designed to mimic the presumed bicyclic transition state. A truly potent inhibitor has eluded our grasp, however, as reflected by the fact that most compounds investigated bind more weakly to chorismate mutase/prephenate dehydrogenase than does chorismate (as reflected by the substrate Km value). 1-Adamantyl phosphonic acid is the most potent inhibitor previously reported, with an I50 value twenty-fold less than chorismate Km under comparable conditions. 

These materials were examined as inhibitors of the Escherichia coli chorismate mutase/prephenate dehydrogenase however, none is a particularly potent inhibitor (Table 2). Although the oximino derivative E-39 has many of the features of our desired target 36, the N-hydroxyl group is not ionized at neutral pH (pK " 12) and it is only a poor carboxylate mimic. 

I would like to express my appreciation to Loretta A. McQuaid, Paul M. Chouinard, Charles R. Johnson, and Robert T. Lum, who carried out the research described above. We would like to thank Professor Jeremy R. Knowles for providing us with purified chorismate mutase-prephenate dehydrogenase used in the studies described in Tables 2 and 3 as well as an authentic sample of shikimate-3-phosphate. The financial support of Merck Sharp Dohme, as well as the National Institutes of Health (grant no. GM-28965), is also gratefully acknowledged. 

L-Tyrosine biosynthesis starts with the condensation of phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), the intermediates of the glycolytic pathway and pentose phosphate pathway, respectively, which is catalyzed by 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS aroE/aroG/aroH). The resultant 3-deoxy-D-arabino-heptulosonate (DAHP) is converted into chorismate through the shikimate pathway with seven reactions. In plants, prephenate (PPA) is converted into L-arogenate by transamination whereas in E. coli, PPA is converted to p-hydroxyphenylpyruvate (HPP) by prephenate dehydrogenase, which is a bifunctional enzyme that behaves as chorismate mutase/prephenate... 

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