Chorismate-prephenate

Figure 11 Chorismate-prephenate rearrangement catalyzed by antibody 1F7 raised against a bicyciic hapten that mimics the chair conformation of the transition state of the reaction and X-ray structure of the corresponding antibody 1 F7-hapten compiex.

Wiest, O. Honk, K. N. Stabilization of the transition state of the chorismate-prephenate rearrangement An ab initio study of enzyme and antibody catalysis, J. Am. Chem. Soc. 1995,117, 11628-11639. 

Epoxide opening by benzeneselenolate anion gave the rphenyl selenide with high regioselectivity (Table 8, entry 4)22. Oxidation and rearrangement yielded (+)- ra/t. -2-cyclohexene-1,4-diol. A similar approach was the key step in the synthesis of a ( + )-chorismate-prephenate analog (Table 8, entry 5)24. 

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. 

However, recent studies with bacteria other than E. coli and the availability and analysis of genomic sequences from many prokaryotic and eukaryotic microorganisms have established that there are three different routes to the soluble intermediate 4-HB (Figure 12). The three routes to 4-HB are (1) the CPL reaction, (2) the tyrosine-4-hydroxyphenylpynivate (THP) pathway, and (3) the chorismate-prephenate- 4-hydroxyphenylpyruvate (CPHP) pathway. 

J.A. Connelly and D.L. Siehl, Purification of Chorismate, Prephenate, and Arogenate by HPLC, Methods in Enzymology, 142(1987)422. 

A Try mutant would not be subject to feedback inhibition by overproduction of tryptophan. Also, the mutation may allow more chorismate to proceed to prephenate via E3 (see Figure 8.4) and thus through to L-phenylalanine. 

Figure 1. Schematic outline of various products and associated enzymes from the shikimate and phenolic pathways in plants (and some microorganisms). Enzymes (1) 3-deoxy-2-oxo-D-arabino-heptulosate-7-phosphate synthase (2) 5-dehydroquinate synthase (3) shikimate dehydrogenase (4) shikimate kinase (5) 5-enol-pyruvylshikimate-3-phosphate synthase (6) chorismate synthase (7) chorismate mutase (8) prephenate dehydrogenase (9) tyrosine aminotransferase (10) prephenate dehydratase (11) phenylalanine aminotransferase (12) anthranilate synthase (13) tryptophan synthase (14) phenylalanine ammonia-lyase (15) tyrosine ammonia-lyase and (16) polyphenol oxidase. (From ACS Symposium Series No. 181, 1982) (62).

In this contribution, we describe work from our group in the development and application of alternatives that allow the explicit inclusion of environment effects while treating the most relevant part of the system with full quantum mechanics. The first methodology, dubbed MD/QM, was used for the study of the electronic spectrum of prephenate dianion in solution [18] and later coupled to the Effective Fragment Potential (EFP) [19] to the study of the Claisen rearrangement reaction from chorismate to prephenate catalyzed by the chorismate mutase (CM) enzyme [20]. 

The Shikimate pathway is responsible for biosynthesis of aromatic amino acids in bacteria, fungi and plants [28], and the absence of this pathway in mammals makes it an interesting target for designing novel antibiotics, fungicides and herbicides. After the production of chorismate the pathway branches and, via specific internal pathways, the chorismate intermediate is converted to the three aromatic amino acids, in addition to a number of other aromatic compounds [29], The enzyme chorismate mutase (CM) is a key enzyme responsible for the Claisen rearrangement of chorismate to prephenate (Scheme 1-1), the first step in the branch that ultimately leads to production of tyrosine and phenylalanine. 

Scheme 1-1. Transition state for the conversion of chorismate into prephenate. Also indicated are the Glu78 and Arg90 residues from chorismate mutase...

Figure 1-4. Energy profiles for the reaction of chorismate to prephenate. (a) Profile in vacuum for the forward (squares) and reverse (filled circles) reactions, (b) Profiles for forward reaction in water (filled circles), and in the enzyme with only the substrate in the QM zone (squares) and with substrate plus chorismate mutase side chains glu78 and arg90 in the QM zone (diamonds)...

Figure 1-5. Free energy profile for the reaction from chorismate (RC 1.75) to prephenate (RC — 1.75), obtained using MSMD and Jarzynski s equality and pulling speeds of 2.0 A/ps (red) and 1.0 A/ps (green), and using umbrella sampling (blue)...

Chorismate mutase catalyzes the Claisen rearrangement of chorismate to prephenate at a rate 106 times greater than that in solution (Fig. 5.5). This enzyme reaction has attracted the attention of computational (bio)chemists, because it is a rare example of an enzyme-catalyzed pericyclic reaction. Several research groups have studied the mechanism of this enzyme by use of QM/MM methods [76-78], It has also been studied with the effective fragment potential (EFP) method [79, 80]. In this method the chemically active part of an enzyme is treated by use of the ab initio QM method and the rest of the system (protein environment) by effective fragment potentials. These potentials account... 

Schultz and coworkers (Jackson et a ., 1988) have generated an antibody which exhibits behaviour similar to the enzyme chorismate mutase. The enzyme catalyses the conversion of chorismate [49] to prephenate [50] as part of the shikimate pathway for the biosynthesis of aromatic amino acids in plants and micro-organisms (Haslam, 1974 Dixon and Webb, 1979). It is unusual for an enzyme in that it does not seem to employ acid-base chemistry, nucleophilic or electrophilic catalysis, metal ions, or redox chemistry. Rather, it binds the substrate and forces it into the appropriate conformation for reaction and stabilizes the transition state, without using distinct catalytic groups. 

Table 1 Kinetic and thermodynamic parameters for the spontaneous, enzyme-catalysed and antibody-catalysed conversion of chorismic acid [23] into prephenic acid [24],...

Claisen rearrangement chorismic acid to prephenic acid... 

Aryl side chain containing L-a-amino acids, such as phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), are derived through the shikimate pathway. The enzymatic transformation of phosphoenolpyr-uvate (PEP) and erythro-4-phosphate, through a series of reactions, yields shikimate (Scheme 2). Although shikimate is an important biosynthetic intermediate for a number of secondary metabolites, this chapter only describes the conversion of shikimate to amino acids containing aryl side chains. In the second part of the biosynthesis, shikimate is converted into chorismate by the addition of PEP to the hydroxyl group at the C5 position. Chorismate is then transformed into prephenate by the enzyme chorismate mutase (Scheme 3). 

The antibodies can also act like entropy traps by stabilizing a particular conformation of a substrate that is favorable to the formation of the TS. It is the case of the antibody 1F7 catalyzing the transformation of chorismate into prephenate," which stabilizes, thanks to several hydrogen bonds and an ionic bond between an arginine (Arg H95) and a carboxylate substituent of the substrate, the conformation of the chorismate which will give rise to the TS in a chair conformation for this reaction (Figure 11). 

This enzyme [EC 5.4.99.5] catalyzes the interconversion of chorismate and prephenate. 

This enzyme [EC 4.2.1.51] catalyzes the conversion of prephenate to phenylpyruvate, water, and carbon dioxide. This enzyme in enteric bacteria also possesses a chorismate mutase activity and converts chorismate into prephenate. 

Figure 1. Hypothetical mechanism for shuttling of intermediates of the common aromatic pathway between plastidic and cytosolic compartments. Enzymes denoted with an asterisk (DAHP synthase-Co, chorismate mutase-2, and cytosolic anthranilate synthase) have been demonstrated to be isozymes located in the cytosol. DAHP molecules from the cytosol are shown to be shuttled into the plastid compartment in exchange for EPSP molecules synthesized within the plastid. Abbreviations C3, phosphoenolpyruvate C4, erythrose 4-P DAHP, 3-deoxy-D-arabino-heptulosonate 7-phosphate EPSP, 5-enolpyruvylshikimate 3-phosphate CHA, chorismate ANT, anthranilate TRP, L-tryptophan PPA, prephenate AGN, L-arogenate TYR, L-tyrosine and PHE, L-phenylalanine.

The hydrophobic effects between the apolar gronps involved in the Diels-Alder reaction also occnr when the apolar gronps belong to the same molecules, and thus should also be beneficial to the Claisen rearrangement. The nonenzymatic rearrangement of chorismate to prephenate occurs 100 times faster in water than in methanol (Copley and Knowles, 1987 Grieco et al., 1989). 

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