Mutagenesis Study of Active Site Residues in Chorismate Mutase from Bacillus subtilis

Abstract

Chorismate mutase catalyses the [3,3] Claisen rearrangement of chorismate (1) to prephenate (2), the committed step in the biosynthesis of tyrosine and phenylalanine in bacteria, fungi, and higher plants 1 (Scheme 1). Despite more than two decades of studies on this novel biological rearrangement, 2-15 the mechanism of the enzyme-catalyzed reaction remains unclear. Substrate labeling 5,7 and kinetic isotope effect studies 6 demonstrated that both the uncatalyzed and catalyzed reactions proceed through a chairlike transition state in which the C5-O7 bond cleavage precedes C9-C1 bond formation. Mechanistic proposals which have been suggested previously include protonation or deprotonation of the C4 hydroxyl by an active site general acid or base, respectively, nucleophilic catalysis by attack of an active site nucleophile at C5, and simple conformational restriction of the substrate. 9,14 Recently, X-ray crystal structures of monofunctional chorismate mutases from Bacillus subtilis (BsCM) 16,17 Escherichia coli (EcCM), 18 and catalytic antibody 1F7 19 bound to the endo-oxabicyclic transition state analogue 3 11 were solved (Figure 1). Analysis of the active site structures has led to a general mechanistic hypothesis that the enzymes and antibody stabilize the chairlike transition state geometry via a series of electrostatic and hydrogen-bonding interactions, 17-20 which is consistent with the earlier finding that the rearrangement of chorismate and related compounds is more facile in hydrogen-bonding solvents. 8,10 In addition, it has been speculated that enzyme active site residues may stabilize the developing charge on the enol ether oxygen and the cyclohexadiene ring in the polar transition state. 6,10 The availability of the structures combined with the ability to mutate specific residues provides an opportunity to test these mechanistic hypotheses and identify key catalytic residues. We have generated and characterized a series of 16 mutants of the B. subtilis enzyme.The gene encoding BsCM was subcloned from plasmid pBSCM2 21 into the phagemid pAED4 22 under the control of the T7 RNA polymerase promoter. Six histidines were added to the carboxy terminus using PCR 23 to afford plasmid pCM6XH. Mutant genes were constructed using the method of Kunkel, 24 and the resulting proteins were expressed in E. coli strain BL21 which carries the gene for T7 RNA polymerase behind the lacUV5 promoter. 25 Mutant proteins were purified to homogeneity (as determined by SDS-PAGE and Coomassie staining) in a single step using IMAC affinity chromatography on Ni(II)-chelating resin (Novagen). 26,27 The structural integrity of the mutants was assayed by circular dichroism (CD) spectroscopy. The CD spectra of all mutants between 200 and 260 nm were superimposable with that of the wild-type (wt) protein, suggesting that no significant structural changes were caused by the mutations. Activities of the mutants were determined by monitoring the disappearance of chorismate spectrophotometrically at 274 or 304 nm (∆ 274 ) 2340 M -1 cm -1 ...

Electrostatic transition state stabilization rather than reactant destabilization provides the chemical basis for efficient chorismate mutase catalysis

“…Together with previous mutagenesis (26)(27)(28) and modeling studies (23,24,37), our results provide a unified view of CM catalysis. They highlight the importance of a cationic residueeither an arginine or a lysine-proximal to the ether oxygen of the substrate in the transition state.…”

“…1B) with a noncharged arginine analog, the amino acid citrulline, which is not normally found in proteins. Consistent with the hypothesis that Arg90 stabilizes a developing partial negative charge in the transition state (18,(26)(27)(28), the Arg90Cit substitution has little effect on the ground state Michaelis complex, judging from the mere threefold increase in K m for chorismate (note that K m approximates K s for WT BsCM) (29), but it greatly impairs catalytic efficiency, reducing both k cat and k cat /K m by more than 4 orders of magnitude (25). These changes suggest that the positively charged arginine contributes up to 5.9 kcal/mol to transition state stabilization but only 0.6 kcal/mol to the binding energy of the ground state.…”

Chorismate‐Mutase‐Catalyzed Claisen Rearrangement

“…To identify the key residues that play an important role in the catalysis, a number of active site mutants were generated and characterized for EcCM [27,35], yeast CM [28] and BsCM [25,29,31,36] and the effects of mutations on the activity have been determined. For EcCM, Arg28, Arg11′ and Lys39 are involved in the direct interactions with the two carboxylate groups as well as the ether oxygen of TSA in the X-ray structure (Scheme 1.4a).…”

Quantitative evaluation of noncovalent chorismate mutase-inhibitor binding by ESI-MS

“…For the wild type enzyme BsCM and the TSA inhibitor a K d of 1.7 Ϯ 1 M was determined, which compares well with the value of 1.0 Ϯ 0.2 M determined in the solutionphase assay. In the literature values between 1.7 [26] and 3 M [16] have been reported. For the truncated enzyme BsCM 1-120 and TSA we determined K d to be 37 Ϯ 6 M with our mass spectrometric method whereas a standard solution assay gave 25 Ϯ 1.7 M. Again, the K d values determined by mass spectrometry and the solution-phase assay are in good agreement.…”