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Functional identification of the general acid and base in the dehydration step of indole-3-glycerol phosphate synthase catalysis.

ABSTRACT: The tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase is a proposed target for new antimicrobials and is a favored starting framework in enzyme engineering studies. Forty years ago, Parry proposed that the enzyme mechanism proceeds through two intermediates in a series of condensation, decarboxylation, and dehydration steps. X-ray crystal structures have suggested that Lys-110 (numbering according to the Sulfolobus solfataricus enzyme) behaves as a general acid both in the condensation and dehydration steps, but did not reveal an efficient pathway for the reprotonation of this critical residue. Our mutagenesis and kinetic experiments suggest an alternative mechanism whereby Lys-110 acts as a general acid in the condensation step, but another invariant residue, Lys-53, acts as the general acid in the dehydration step. These studies also indicate that the conserved residue Glu-51 acts as the general base in the dehydration step. The revised mechanism effectively divides the active site into discrete regions where the catalytic surfaces containing Lys-110 and Lys-53/Glu-51 catalyze the ring closure (i.e. condensation and decarboxylation) and dehydration steps, respectively. These results can be leveraged toward the development of novel inhibitors against this validated antimicrobial target and toward the rational engineering of the enzyme to produce indole derivatives that are highly prized by the pharmaceutical and agricultural industries.

SUBMITTER: Zaccardi MJ

PROVIDER: S-EPMC3772182 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Functional identification of the general acid and base in the dehydration step of indole-3-glycerol phosphate synthase catalysis.

Zaccardi Margot J MJ Yezdimer Eric M EM Boehr David D DD

The Journal of biological chemistry 20130730 37

The tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase is a proposed target for new antimicrobials and is a favored starting framework in enzyme engineering studies. Forty years ago, Parry proposed that the enzyme mechanism proceeds through two intermediates in a series of condensation, decarboxylation, and dehydration steps. X-ray crystal structures have suggested that Lys-110 (numbering according to the Sulfolobus solfataricus enzyme) behaves as a general acid both in the cond ...[more]

PMID: 23900843

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Project description:Substrate binding, product release, and likely chemical catalysis in the tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase (IGPS) are dependent on the structural dynamics of the ?1?1 active-site loop. Statistical coupling analysis and molecular dynamic simulations had previously indicated that covarying residues in the ?1?1 and ?2?2 loops, corresponding to Arg54 and Asn90, respectively, in the Sulfolobus sulfataricus enzyme (ssIGPS), are likely important for coordinating functional motions of these loops. To test this hypothesis, we characterized site mutants at these positions for changes in catalytic function, protein stability and structural dynamics for the thermophilic ssIGPS enzyme. Although there were only modest changes in the overall steady-state kinetic parameters, solvent viscosity and solvent deuterium kinetic isotope effects indicated that these amino acid substitutions change the identity of the rate-determining step across multiple temperatures. Surprisingly, the N90A substitution had a dramatic effect on the general acid/base catalysis of the dehydration step, as indicated by the loss of the descending limb in the pH rate profile, which we had previously assigned to Lys53 on the ?1?1 loop. These changes in enzyme function are accompanied with a quenching of ps-ns and µs-ms timescale motions in the ?1?1 loop as measured by nuclear magnetic resonance studies. Altogether, our studies provide structural, dynamic and functional rationales for the coevolution of residues on the ?1?1 and ?2?2 loops, and highlight the multiple roles that the ?1?1 loop plays in IGPS catalysis. Thus, substitution of covarying residues in the active-site ?1?1 and ?2?2 loops of indole-3-glycerol phosphate synthase results in functional, structural, and dynamic changes, highlighting the multiple roles that the ?1?1 loop plays in enzyme catalysis and the importance of regulating the structural dynamics of this loop through noncovalent interactions with nearby structural elements.

Dataset Information

Functional identification of the general acid and base in the dehydration step of indole-3-glycerol phosphate synthase catalysis.

Publications

Functional identification of the general acid and base in the dehydration step of indole-3-glycerol phosphate synthase catalysis.

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