Chorismate mutase

Chorismate mutase provides an example of an enzyme where QM/MM calculations have identified an important catalytic principle at work [8], This enzyme catalyses the Claisen rearrangement of chorismate to prephenate. The reaction within the enzyme is not believed to involve chemical catalysis, and this pericylic reaction also occurs readily in solution. Lyne et al. [8] investigated the reaction in chorismate mutase in QM/MM calculations, at the AMI QM level (AMI was found to perform acceptably well for this reaction in comparisons with ab initio results for the reaction in the gas phase [8]). Different sizes of QM system were tested in the QM/MM studies (e.g. including the substrate and no, or up to three, protein side chains), and similar results found in all cases. The reaction was modelled by minimization along an approximate reaction coordinate, defined as the ratio of the forming C-C and breaking C-0 bonds. Values of the reaction coordinate were taken from the AMI intrinsic reaction coordinate for the gas-phase reaction. 

The calculated barrier to reaction in chorismate mutase was 17.8 kcal/mol, compared to 42 kcal/mol in the gas phase. Factors other than substrate distortion also play an important part in reducing the barrier to reaction in the enzyme important interactions were identified by a simple decomposition analysis (as described in sections 6.1 and 6.2 above). It was found that Glu78 and Arg90 specifically stabilize the transition state, relative to the bound substrate [8]. Overall, therefore, catalysis in chorismate mutase can be rationalized in terms of a combination of substrate strain and transition state stabilization. While it is possible to analyse all these catalytic effects as arising from maximal binding in the enzyme being achieved at the transition state, it appears useful to separate the different types of contribution. The possible role of substrate destabilization/distortion or strain in lowering the barrier to reaction in enzyme reactions, as put forward by Haldane [219], and invoked in 

QM/MM methods do require some care in their application, for example in the choice of QM system, and other practical aspects discussed in section 5, and the nature of the QM/MM partitioning and interactions, as outlined in section 2 above. Their application is not, as yet, as standardized as for purely MM or purely QM methods. For any given application, the performance of the QM/MM model should be tested. They are necessarily hybrid methods, and it is not always clear beforehand which coupling schemes may be most appropriate, and consistent with both the QM method and MM force field. Testing of different approaches for QM/MM combination will therefore continue to be important. Possible areas for improvement include the treatment of the QM/MM junction in partitioned covalently bonded molecules, and MM representations going beyond the simple invariant point charge model. 

Methods to reduce the computational demand of QM/MM simulations are also being developed, including mapping from simulations run with more 

The author would like to thank his collaborators in the work described here. 

The conversion of chorismate into prephenate occurs at a critical point in the shikimic acid pathway the biosynthesis of a variety of aromatics branch off from here. Since CM appears in lower organisms (such as fungi and bacteria) and not in mammals, it is an excellent target for the development of antibacterial and antifungal agents. 

In addition to its biochemical importance, CM has drawn attention from chemists looking to study enzyme activity for three primary reasons. First, the substrate 1 binds to the enzyme CM withont forming any covalent linkages, so that major electronic reorganizations do not need to be considered. Second, unlike many enzyme-catalyzed reactions, the reaction mechanism for the conversion of 1 into 2 is the same in both the enzyme environment and in solution in the absence of the enzyme. Last, the kinetics of the enzyme activity are well known, with CM increasing the rate of the reaction by 10 over the rate in aqueous solution. This corresponds to a reduction in the activation enthalpy of 20.7 0.4 kcal moL in solution to 15.9 kcal mol in the enzymatic environment. The activation entropy is -12.9 0.4 eu in solution but is reduced to essentially nil in the enzyme. In other words, AG = 24.5 kcal moL in solution but only 15.4 kcal mol in CM.  

A number of research groups have employed QM/MM methods toward understanding the activity of CM, engendering a small controversy. Bmice has argned that CM exhibits a classic example of the involvement of near attack conformers (NAC). Bmice s contention is that CM aids in the formation of the diaxial conformation, thus increasing the population of the NAC and thereby accelerating the reaction. 

The NAC is the arrangement (or a gronp of arrangements) of reactants in an appropriate geometry to facihtate passing Ihrongh the TS. So, in the case of Reaction 9.4, the NAC is the collection of molecnles in a diaxial conformation that can then proceed through the TS and on the prephenate. Some have interpreted this as 

Bruice s contention that there is very little stabilization of the TS by CM, rather it is a favorable binding of the NAC that accounts for its catalytic activity, has been met with much skepticism. An early study by Wiest and Houk looked at chorismate models with coordinated waters and aminidium cations in positions that matched interacting groups found in the crystal structure of CM with a coordinated substrate. These model computations suggested that the neighboring amino acid residues could be stabilizing the TS. 

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Chorismate Mutase catalyzed Claisen Rearrangement- 10 rate enhancement over non-enzymatic reaction... 

T.-phenyl alanine C. glutamicum C. glutamicum aro F, chorismate mutase, PRDH 28 ... 

Chorismate mutase Proprietary 15 K (a) UNITY, (b) FlexX 4 of 15 tested [119]... 

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).

The enzyme chorismate mutase was found to accelerate the Claisen rearrangement of chorismic acid.147 For many years, the origin of the acceleration perplexed and intrigued chemists and biochemists. Polar... 

The differences in the rate constant for the water reaction and the catalyzed reactions reside in the mole fraction of substrate present as near attack conformers (NACs).171 These results and knowledge of the importance of transition-state stabilization in other cases support a proposal that enzymes utilize both NAC and transition-state stabilization in the mix required for the most efficient catalysis. Using a combined QM/MM Monte Carlo/free-energy perturbation (MC/FEP) method, 82%, 57%, and 1% of chorismate conformers were found to be NAC structures (NACs) in water, methanol, and the gas phase, respectively.172 The fact that the reaction occurred faster in water than in methanol was attributed to greater stabilization of the TS in water by specific interactions with first-shell solvent molecules. The Claisen rearrangements of chorismate in water and at the active site of E. coli chorismate mutase have been compared.173 It follows that the efficiency of formation of NAC (7.8 kcal/mol) at the active site provides approximately 90% of the kinetic advantage of the enzymatic reaction as compared with the water reaction. 

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. 

Besides the obvious biological interest, chorismate mutase is important for being a rare example of an enzyme that catalyses a pericyclic reaction (the Claisen rearrangement), which also occurs in solution without the enzyme, providing a unique... 

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)...

Lambert, K.N., Allen, K.D. and Sussex, I.M. (1999) Cloning and characterization of an esophageal-gland-specific chorismate mutase from the phytoparasitic nematode Meloidogyne javanica. Molecular Plant-Microbe Interactions 12, 328-336. 

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... 

The partitioning of the system in a QM/MM calculation is simpler if it is possible to avoid separating covalently bonded atoms at the border between the QM and the MM regions. An example is the enzyme chorismate mutase [39] for which the QM region could include only the substrate, because the enzyme does not chemically catalyze this pericyclic reaction. In studies of enzyme mechanisms, however, this situation is exceptional, and usually it will be essential, or desirable, to include parts of the protein (for example catalytic residues) in the QM region of a QM/MM calculation, i.e. the boundary between the QM and MM regions will separate covalently bonded atoms (Fig. 6.1). 

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. 

The conversion of [49] into [50] involves a Claisen rearrangement. Once this was realized it was less surprising that no specific catalytic groups on the enzyme are involved. Support for the Claisen-type mechanism comes from the inhibition shown by the bicyclic dicarboxylate [51], prepared by Bartlett and Johnson (1985) as an analogue of the presumed transition state [52], This same structure [51], coupled through the hydroxyl group to a small protein, was used as a hapten to induce antibodies, one (out of eight) of which mimics the behaviour of chorismate mutase, albeit less efficiently (Table 7). 

Table 7 Chorismate mutase and a catalytic antibody mimic."...

Table 8 Another catalytic antibody mimic of chorismate mutase."...

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). 

Fig. 5. Comparison of suppression efficiencies of five tRNAs A in T4 lysozyme at site 82, and B in chorismate mutase at site 88. Suppression efficiencies are defined as the amount of full-length protein divided by the sum of the full-length and truncated protein produced in each reaction. The suppression efficiencies shown represent the average of two trials. The tRNAs are identified below each bar Y yeast, E E. coli T Tetrahymena rt readthrough (un-acylated tRNA) V acylated with valine hE acylated with homoglutamate. Reprinted with permission [33]...

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. 

BUTYRYLCHOLINE ESTERASE CHOLINE SULFATASE CHOLINE SULFOTRANSFERASE CHOLOYL-OoA SYNTHETASE OHONDROITIN 4-SULFOTRANSFERASE OHONDROSULFATASES CHORISMATE MUTASE CHORISMATE SYNTHASE Chromatin self-assembly,... 

Krengel, U., Dey, R., Sasso, S., Okvist, M., Ramakrishnan, C. and Kast, P. (2006). Preliminary X-ray crystallographic analysis of the secreted chorismate mutase from Mycobacterium tuberculosis a tricky crystallization problem solved. Acta Crystallogr. F 62, 441 45. 

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.

Of the separately compartmented isozyme pairs that exist for DAHP synthase, chorismate mutase, and anthranilate synthase, each isozyme member of a given pair has different properties of regulation and other distinctive characteristics (see Tables I and II). This suggests a high probability that each isozyme is the gene product of a different gene. 

Table II. Differential Properties of Chorismate Mutase Isozymes...

To what extent is the response of cytosolic and plastidic isozymes of the shikimate pathway coordinated or coupled with one another and to alterations in expression of enzymes of the flavonoid and phenylpropanoid-pathway segments Some of the emerging information is given in Figure 6. Thus, light induction, well known to induce PAL and enzymes of the flavonoid pathway, also induces both DS-Mn and DS-Co in parsley cell cultures (49). However, only the cytosolic CM-2 (and not the plastidic CM-1) was induced. Fungal elicitor was reported to induce only DS-Mn—not DS-Co or either of the chorismate mutase isozymes (49). Previous studies... 

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