Background: Bacteria require the enzyme indole-3-glycerol phosphate synthase for the production of Trp. Results: Glu-51 and Lys-53 are identified as the base and acid acting in the dehydration step of enzyme catalysis. Conclusion: Ring closure and dehydration steps are catalyzed by distinct active-site surfaces. Significance: Enzyme inhibitors targeted against these active-site surfaces may serve as novel antibiotics.

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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 b1a1 active-site loop. Statistical coupling analysis and molecular dynamic simulations had previously indicated that covarying residues in the b1a1 and b2a2 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 b1a1 loop. These changes in enzyme function are accompanied with a quenching of ps-ns and ms-ms timescale motions in the b1a1 loop as measured by nuclear magnetic resonance studies. Altogether, our studies provide structural, dynamic and functional rationales for the coevolution of residues on the b1a1 and b2a2 loops, and highlight the multiple roles that the b1a1 loop plays in IGPS catalysis. Thus, substitution of covarying residues in the active-site b1a1 and b2a2 loops of indole-3-glycerol phosphate synthase results in functional, structural, and dynamic changes, highlighting the multiple roles that the b1a1 loop plays in enzyme catalysis and the importance of regulating the structural dynamics of this loop through noncovalent interactions with nearby structural elements.

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