The Claisen Rearrangement of an Unusual Substrate in Chorismate

Sharon E. Worthington*, and Morris Krauss. Center Advanced Research in Biotechnology, 9600 Gudelsky Drive, Rockville, Maryland 20850. J. Phys. Chem...
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J. Phys. Chem. B 2001, 105, 7096-7098

The Claisen Rearrangement of an Unusual Substrate in Chorismate Mutase Sharon E. Worthington* and Morris Krauss Center AdVanced Research in Biotechnology, 9600 Gudelsky DriVe, RockVille, Maryland 20850 ReceiVed: January 22, 2001

The calculated reaction path for an unusual substrate of chorismate mutase (Bacillus subtilis) is found to be completely comparable to that of the native chorismate. In the unusual substrate, the cyclohexadienyl ring in chorismate is replaced by dihydropyridine. Previous theoretical calculations of the native reaction path provide the basis for predicting that the interactions of the unusual reactant in the active site are electronically analogous to the native. Three unusual substrates are obtained by replacing each of the C-H moieties in the cyclohexadienyl ring by a nitrogen atom. This substitution does not substantially alter the electronic characteristics of the ring both with regard to the catalytic activation by the active site and the interaction with hydrophobic groups in the active site. The activation energies and optimized structures along the reaction path are obtained for the tautomers of the unusual reactant and product. The possibility of ultimately obtaining an unusual amino acid by this pathway is discussed.

1. Introduction In bacteria, plants, and fungi, aromatic amino acids are synthesized from a central molecule in the shikimate pathway, chorismate.4 There are several pathways for the conversion of chorismate into L-phenylalanine and L-tyrosine, depending on the organism. All require the production of prephenate by the Claisen rearrangement of chorismate catalyzed by chorismate mutase. Recently, we have determined the reaction path for this rearrangement in the active site of chorismate mutase.10 In addition to the calculation of the activation energy, the structures of the reactant and transition state were determined in the enzyme active site. From analysis of these structures and the electronic characteristics of the active site, we determined the catalytic properties of the enzyme. Two residues, glu78 and tyr108, were identified as catalytically significant in chorismate mutase from Bacillus subtilis. Although the active site is very ionic, these ionic or polar residues bind directly to the carboxylate groups, the ether bond, and the C4-OH substituent on the ring. The cyclohexadienyl ring interacts mostly with hydrophobic residues primarily through dispersive forces. We suggest that conservative modifications in the ring can be made that do not alter the electronic characteristics of the ring or its interactions with the hydrophobic residues substantially. Recently, the development of a new combined molecular dynamics (MD) and quantum chemical (QM) methodology has been employed to study enzyme systems. In this work, the versatility of this new method is demonstrated by the study of a non-native substrate for chorismate mutase. In the MD/QM methodology, the reaction path is calculated for a catalytically competent arrangement of the protein. A catalytically competent arrangement is defined as the protein field for which the reaction occurs with the activation energy equal to or less than the experimentally observed activation energy. A number of essentially different protein environments may support comparable reaction path energetics. These environments or motifs can be ascertained from X-ray structures or molecular dynamics simulations. Analysis of the quantum calculation of the reaction path for these conformations or motifs provides essential insight into the electronic characteristics of the enzyme reaction. These

findings are tested both by effect of mutations and the use of an unusual substrate for the reaction. We have chosen to explore what is perhaps the simplest modification, where the cyclohexadienyl ring is converted into a dihydropyridinyl ring by altering in turn a C-H group into an N atom, producing three dihydropyridine analogues of chorismate, as shown in Figure 1. Even though the modification of the ring is simple and obvious, previous experimental changes to the substrate have not involved the cyclohexadienyl ring. Three substrate analogues are possible with N atoms at positions 2, 5, and 6. The numbering in Figure 1 is consistent with the literature pertaining to chorismate in order to compare this work easily with previous studies on the native chorismate mutase reaction. However, the proper IUPAC name for the N5 tautomer (C in Figure 1) is (6S,5R)-5-(1-carboxtvinyloxy)-6-hydroxy-5,6dihydropyridine-3-carboxylic acid. Reaction paths for the three unusual substrates have been calculated by the same methodology used for the native reaction.9 2. Method and Results Calculation of the reaction path and transition state requires that ab initio quantum chemistry be used. The large number of atoms in the complex of the active site and reactant would normally render such a calculation intractable unless we treat those atoms in the active site not involved in the chemistry differently from the reacting complex. Effective fragment potentials (EFPs) represent the interactions of all groups in the active site that are only involved in nonchemical binding.3 The EFPs replace the exchange repulsion, electrostatic potential, polarization, and constrained charge transfer from the spectator region in the quantum Hamiltonian of the total complex. The effective fragment potential (EFP) method is implemented in the GAMESS program suite.6 The electrostatic and polarization components of the EFP for a residue are calculated within GAMESS, but the repulsive EFPs describing the amino acid residues have been determined separately.10 Using these EFPs, we have obtained optimized structures of enzyme-substrate complexes at the Hartree-Fock (HF) level of theory. All restricted Hartree-Fock (RHF) calculations are done with

10.1021/jp010228o CCC: $20.00 © 2001 American Chemical Society Published on Web 06/19/2001

Claisen Rearrangement in Chorismate Mutase

J. Phys. Chem. B, Vol. 105, No. 29, 2001 7097 TABLE 1: Reactant Bond Distances and Hydrogen Bond Distances (Å)a bond

CM (A)

CM (B)

CM (C)

CM (D)

C3-O7 C1-C9 C4-O12 O12-H R90-O7 E78-H

1.505 3.644 1.437 0.983 2.601 1.864

1.486 3.418 1.433 0.985 2.628 1.831

1.495 3.617 1.424 0.982 2.577 1.936

1.501 3.660 1.432 0.986 2.552 1.823

a (A) Chorismate, (B) C2 to N2 substitution, (C) C5 to N5 substitution, and (D) C6 to N6 substitution.

TABLE 2: Transition-State Bond Distances and Hydrogen Bond Distances (Å)a

Figure 1. Numbered structures of chorismate (A) and the three tautomers (B, C, and D) of the dihydropyridinyl ring substrate

effective core potentials and their concomitant basis sets. At the HF geometries, the single-point energies are obtained at the second-order perturbation, MP2, level. In this study, all calculations are done with the 4-31G SBK basis set.7,8 Stationary points have been determined for the reactant/active site and transition-state (TS)/active-site complexes. The active site conformation is determined from the X-ray structure of a transition state analogue bound in chorismate mutase from B. subtilis. The experimental binding of the endo-oxabicyclic transition state analogue (TSA) is found in the Protein Data Bank (PDB)1 as 2CHT.2 All calculations of substrate binding and reactive behavior are initiated from the crystal structure of the active site situated at the A/B interface with bound TSA. The model active site is comprised of the ionic and polar amino acid residues lys60, arg63, and cys75 from monomer A and arg7, glu78, met79, arg90, and tyr108 from monomer B. The nonpolar residue, phe57, from monomer A is also included in the active site model. Since we identify glu78 as chemically involved through charge transfer interactions to the ring, this residue is included in the all-electron part of the calculation. The fundamental assumption is that the protein environment is fixed during the reaction, which is very fast relative to the time taken to achieve the catalytically competent conformation. The reaction path can be calculated within the protein field derived from the TSA complex that is suitable for a reactive conformation. In this note, we will not attempt to generate other catalytically competent conformations or quantum motifs by prior molecular dynamics simulations. The present calculations will show the reactivity of the dihydropyridine substrates in chorismate mutase. Significant distances that characterize the reactant and TS complexes are shown in Tables 1 and 2, along with the analogous values found for the native chorismate. We have chosen bonds critical for the reaction and for the preactivation: (a) the breaking ether bond, C3-O7, (b) the C1-C9 bond formed in the rearrangement, (c) the C4-O bond of the hydroxyl substituent on the ring, (d) the hydrogen-bond distance from arg90 to the ether oxygen, R90-O7, and (e) the hydrogen-bond distance from glu78 to the hydroxyl proton. A representative substrate/active-site complex for the three substrates is shown

bond

CM (A)

CM (B)

CM (C)

CM (D)

C3-O7 C1-C9 C4-O12 O12-H R90-O7 E78-H

2.351 2.552 1.430 0.993 2.526 1.696

2.144 2.367 1.427 0.992 2.567 1.710

2.209 2.410 1.421 0.991 2.541 1.750

2.275 2.496 1.426 0.996 2.521 1.683

a (A) Chorismate, (B) C to N substitution, (C) C to N substitution, 2 2 5 5 and (D) C6 to N6 substitution.

Figure 2. Optimized structure of the reactant/active site complex for the N (2) substituted tautomer. The substituted nitrogen in the substrate ring is shown in the center of the figure.

in Figure 2. The relative energetics of the three dihydropyridine tautomers were determined in vacuo at the optimized geometries of the substrate-enzyme complexes and are compared below with the analogous values of the substrate tautomers bound in the active site. The in vacuo optimized structures for the tautomers are very different from those embedded in the enzyme and are energetically substantially lower in energy. The activation energies for the three tautomers are compared with the native value in Table 3 with a representative TS/active site structure shown in Figure 3. 3. Discussion The three dihydropyridine tautomers behave almost exactly like chorismate in the Claisen rearrangement. The pyridinyl nitrogen atom does not interact strongly with any of the polar or ionic groups in the active site. As seen in the Figure 2, the

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Worthington and Krauss

TABLE 3: Activation Energies in kcal/mol (kJ/mol)a level of theory in vacuo (1) MP2

9.1 (38.2)

CM (A)

CM (B)

CM (C)

CM(D)

3.9 (16.5) 4.8 (20.1) 6.2 (26.0) 2.6 (10.9)

a (A) Native, (B) C to N substitution, (C) C to N substitution, 2 2 5 5 and (D) C6 to N6 substitution.

Figure 3. Optimized structure of the transition state/active site complex for the N (2) substituted tautomer. The substituted nitrogen in the substrate ring is shown in the center of the figure.

ring is bracketed by the phe57 ring and the cysteine side-chain for all three tautomers. The binding complex of the substituted reactant is essentially the same as the native enzyme; both form the same set of ionic and polar hydrogen bonds. The relative activation energies of the calculated reaction paths are all very similar. The calculated in vacuo total energies of the tautomers at the optimized enzyme conformation differ at most by 8 kcal/ mol between the lowest, N5, and highest energy, N6, tautomers, with the N2 tautomer higher than N5 by 3 kcal/mol. The relative energies of the tautomers in the enzyme active site are on the same order of energy, with N5 being lowest in energy, N2 8 kcal/mol higher, and N6 highest by 11 kcal/mol. The active site environment has a small effect on the binding of the different tautomers. However, the overall conformation is determined by the binding of the carboxylates with the arginine residues and the orientation maintained by the hydrogen bond to tyr108. The comparable activation energies suggest that the active site binds the various tautomers for both the transition states and the reactants. Examination of the figures for the binding of the reactants and their transition states support the conclusion that the tautomers are all bound with the same general conformation and little difference in the binding energetics is to be expected. The electronic activation energies for the dihydropyridine substrates are comparable to the value obtained for chorismate in the native enzyme. The two residues found to be catalytically important in the study of the native enzyme are shown to have the same interactions for these substrates. Preactivation of the reactant ether bond by a glu78 H-bond with the C4-OH moiety on the cyclohexadienyl ring of chorismate was deduced from the distortion in the reactant geometry when bound in the active site. This preactivation of the ether bond is seen in the increased bond lengths found in Table 1 for the reactant complex with

all tautomers. The orientation of tyr108 in the transition states for the unusual tautomers matches that found for the native. Prephenate is central to the production of phenylalanine and tyrosine by conversion first to either phenylpyruvate or arogenate.4 The production of phenylpyruvate begins with dehydration and decarboxylation of prephenate catalyzed by prephenate dehydratase. Phenylpyruvate is then transaminated by phenylpyruvate aminotransferase to yield L-phenylalanine. Little is known about the catalytic mechanism or binding in these enzymes. A recent study could identify only a threonine residue as catalytically important in the prephenate dehydratase enzyme.11 Protonation of the hydroxyl group is the likely step that results in loss of both water and carbon dioxide. Interaction with any other site on the ring is unlikely, but charge transfer through the ring is important. This is analogous to the initial interaction in the rearrangement of chorismate to prephenate, suggesting that the dehydratase catalysis may be active with the dihydropyridine substrates. The subsequent transamination reaction operates on the pyruval moiety, and the phenyl group presumably interacts with hydrophobic groups to provide specificity. The arginine binding to the carboxylate is identified as the primary binding interaction. Depending on the orientation of the pyridinyl ring, there could be a dependence on the tautomer for the activity of the transamination. A crystal structure for the binding of phenyl propionate in an aromatic aminotransferase shows the phenyl ring interacts edge on with a tryptophan and has a long-range interaction with the phosphate of the pyridoxal phosphate cofactor.5 The interaction with the tryptophan or any other hydrophobic residue should be comparable whether the ring was benzyl or pyridinyl. These theoretical calculations suggest that the dihydropyridinyl substrates would have the same activity in chorismate mutase. Examination of the current data suggests that the subsequent reactions in the pathway may be possible for electronically analogous substrates leading to unusual amino acids. Acknowledgment. This work is supported in part by the Advanced Technology Program of the National Institute of Standards and Technology. References and Notes (1) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235-42. (2) Chook, Y. M.; Gray, J. V.; Ke, H.; Lipscomb, W. N. J. Mol. Biol. 1994, 240, 476-500. (3) Day, P. N.; Jensen, J. H.; Gordon, M. S.; Webb, S. P.; Stevens, W. J.; Krauss, M.; Garmer, D.; Basch, H.; Cohen, D. J. Chem. Phys. 1996, 105, 1968-1986. (4) Dewick. Nat. Prod. Rep. 1998, 15, 17-58. (5) Okamoto, A.; Nakai, Y.; Hayashi, H.; Hirotsu, K.; Kagamiyama, H. J. Mol. Biol. 1998, 280, 443-61. (6) Schmidt, M. W.; Baldridge, K.; Boatz, J. A.; Elbert, S. T.; Gordon, M. S.; Jensen, J. H.; Koseki, S.; Matsunaga, N.; Nguyen, K. A.; Su, S.; Windus, T. L.; Dupuis, M.; Montgomery, J. A. J. Comput. Chem. 1993, 14, 1347. (7) Stevens, W. J.; Basch, H., Krauss, M. J. Chem. Phys. 1984, 81, 6026. (8) Stevens, W. J.; Krauss, M.; Basch, H.; Jaisen, P. G. Can. J. Chem. 1992, 70, 612. (9) Worthington, S. E.; Roitberg, A. E.; Krauss, M. J. Phys. Chem. B, submitted for publication. (10) Worthington, S. E.; Krauss, M. Comput. Chem. 2000, 24, 275-285. (11) Zhang S, W. D.; Ganem, B. Biochemistry 2000, 39, 4722-8. (12) Disclaimer: Certain commercial equipment and materials are identified in this paper in order to specify the methods adequately. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.