Differences in the catalytic mechanisms of mesophilic and thermophilic indole-3-glycerol phosphate synthase enzymes at their adaptive temperatures

Zaccardi, Margot J.; Mannweiler, Olga; Boehr, David D.

doi:10.1016/j.bbrc.2012.01.020

Cited by 14 publications

(43 citation statements)

References 21 publications

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“…not diffusion-limited) ( Table 1). This finding is in contrast to what happens at 25°C, at which there is a SVE close to the theoretical maximum of 1, but no SDKIE, and is consistent with product release being rate-determining at this lower temperature (11). It should be noted that pre-steady-state kinetic experiments for ssIGPS also indicated that product release is rate-determining at 25°C, but these experiments were not able to assign microrate constants to each of the individual chemical steps (23).…”

Section: Solvent Viscosity and Isotope Effects Reveal The Rate-determcontrasting

confidence: 55%

“…A, the kinetic mechanism of IGPS. There is a substantial SVE when the rate-determining step is diffusioncontrolled, such as in product release, similar to what occurs at low temperatures (11). A substantial SDKIE is present only when the condensation step is rate-determining (see "Experimental Procedures" and supplemental "Experimental Procedures").…”

Section: Mutational Analysis Suggests That Lys-53 Plays Roles In Bothmentioning

confidence: 94%

“…Protein Overexpression, Purification, and CharacterizationThe overexpression and purification of WT and variant ssIGPS enzymes followed protocols described previously (11,17,18). Steady-state enzyme assays for ssIGPS followed previously established procedures (11,19,20).…”

Section: Methodsmentioning

confidence: 99%

“…Steady-state enzyme assays for ssIGPS followed previously established procedures (11,19,20). In short, ssIGPS activity was measured via fluorescence by monitoring the formation of the product IGP.…”

Section: Methodsmentioning

confidence: 99%

“…Previous mutagenesis experiments have indicated that these residues are important for enzyme catalysis (10, 13) but were not able to assign specific roles for these residues because the kinetic steps that were most affected by these mutations were not elucidated. We have previously used SDKIEs and SVEs to illuminate the rate-determining step(s) for WT ssIGPS under different temperature conditions (11); the SVEs and SDKIEs reported herein are on the steady-state turnover rate constant, k cat . In ssIGPS, the SDKIE reports on the proton transfer event in the condensation reaction (see supplemental "Experimental Procedures"), and the SVE reports on diffusion-controlled events, such as substrate binding and product release.…”

Section: Solvent Viscosity and Isotope Effects Reveal The Rate-determmentioning

confidence: 99%

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Functional Identification of the General Acid and Base in the Dehydration Step of Indole-3-glycerol Phosphate Synthase Catalysis

Zaccardi

Yezdimer

Boehr

2013

Journal of Biological Chemistry

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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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Section: Solvent Viscosity and Isotope Effects Reveal The Rate-determcontrasting

confidence: 55%

Section: Mutational Analysis Suggests That Lys-53 Plays Roles In Bothmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Solvent Viscosity and Isotope Effects Reveal The Rate-determmentioning

confidence: 99%

See 3 more Smart Citations

Functional Identification of the General Acid and Base in the Dehydration Step of Indole-3-glycerol Phosphate Synthase Catalysis

Zaccardi

Yezdimer

Boehr

2013

Journal of Biological Chemistry

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Indole‐3‐Glycerol Phosphate Synthase From Mycobacterium tuberculosis: A Potential New Drug Target

2021

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Tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis, affects millions of people worldwide. Several TB drugs have lost efficacy due to emerging drug resistance and new anti-TB targets are needed. Recent research suggests that indole-3-glycerol phosphate synthase (IGPS) in M. tuberculosis (MtIGPS) could be such a target. IGPS is a (β/α) 8 -barrel enzyme that catalyzes the conversion of 1-(o-carboxyphenylamino)-1deoxyribulose 5'-phosphate (CdRP) into indole-glycerolphosphate (IGP) in the bacterial tryptophan biosynthetic path-way. M. tuberculosis over expresses the tryptophan pathway genes during an immune response and inhibition of MtIGPS allows CD4 T-cells to more effectively fight against M. tuberculosis. Here we review the published data on MtIGPS expression, kinetics, mechanism, and inhibition. We also discuss MtIGPS crystal structures and compare them to other IGPS structures to reveal potential structure-function relationships of interest for the purposes of drug design and biocatalyst engineering.

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Loop‐loop interactions govern multiple steps in indole‐3‐glycerol phosphate synthase catalysis

et al. 2014

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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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Differences in the catalytic mechanisms of mesophilic and thermophilic indole-3-glycerol phosphate synthase enzymes at their adaptive temperatures

Cited by 14 publications

References 21 publications

Functional Identification of the General Acid and Base in the Dehydration Step of Indole-3-glycerol Phosphate Synthase Catalysis

Functional Identification of the General Acid and Base in the Dehydration Step of Indole-3-glycerol Phosphate Synthase Catalysis

Indole‐3‐Glycerol Phosphate Synthase From Mycobacterium tuberculosis: A Potential New Drug Target

Loop‐loop interactions govern multiple steps in indole‐3‐glycerol phosphate synthase catalysis

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