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

Zaccardi, Margot J.; Yezdimer, Eric M.; Boehr, David D.

doi:10.1074/jbc.m113.487447

Cited by 8 publications

(55 citation statements)

References 23 publications

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“…For the R64A/D65A variant, the SVE approaches zero at both temperatures, indicating that IGP release is no longer rate limiting, perhaps due to an increased product off-rate. We have previously noted that the identity of the rate-determining step for ssIGPS can change depending on temperature [24] and amino acid substitutions [25,29]. Such a change in the identity of the rate-determining step implies that the steady-state kinetic parameters underreport changes to the underlying microscopic rate constants.…”

Section: Severing Interactions With the C-terminal Side Of The β1α1 Lmentioning

confidence: 99%

“…Subsequently, Glu51 and Lys53 act as the general base and acid, respectively, in the dehydration step to produce IGP [25]. We have noted that following decarboxylation the intermediate must undergo a rearrangement in order to be properly positioned for dehydration [25]. Although X-ray crystal structures of ssIGPS [25,28] indicate that there is ample room within the active site to accommodate such a rearrangement, motions of ssIGPS, including those of the β1α1 loop, may facilitate this process.…”

Section: Introductionmentioning

confidence: 99%

“…We have noted that following decarboxylation the intermediate must undergo a rearrangement in order to be properly positioned for dehydration [25]. Although X-ray crystal structures of ssIGPS [25,28] indicate that there is ample room within the active site to accommodate such a rearrangement, motions of ssIGPS, including those of the β1α1 loop, may facilitate this process. The β1α1 loop is thus involved in most stages of ssIGPS catalysis, from substrate binding to chemical catalysis, and finally to product release.…”

Section: Introductionmentioning

confidence: 99%

“…In this revised mechanism (Figure 1c), Lys110 (numbering according to ssIGPS) acts a general acid to protonate the C2 carbonyl of CdRP, facilitating ring closure and decarboxylation. Subsequently, Glu51 and Lys53 act as the general base and acid, respectively, in the dehydration step to produce IGP [25]. We have noted that following decarboxylation the intermediate must undergo a rearrangement in order to be properly positioned for dehydration [25].…”

Section: Introductionmentioning

confidence: 99%

“…On the N-terminal side, Asn90 from the β2α2 loop can hydrogen bond with residues on the β1α1 loop, including Arg54. We propose that the β1α1 loop fluctuates between conformations favoring interactions on each side of the loop; (c) chemical mechanism of ssIGPS [25]. Lys110 initiates ring closure and decarboxylation by protonating the carbonyl oxygen.…”

Section: Introductionmentioning

confidence: 99%

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Controlling Active Site Loop Dynamics in the (β/α)8 Barrel Enzyme Indole-3-Glycerol Phosphate Synthase

2016

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Abstract:The β1α1 loop in the tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase (IGPS) is important for substrate binding, product release and chemical catalysis. IGPS catalyzes the ring closure of the substrate 1-(o-carboxyphenylamine)-1-dexoyribulose 5-phosphate to form indole-3-glycerol phosphate, involving distinct decarboxylation and dehydration steps. The ring closure step is rate-determining in the thermophilic Sulfolobus sulfataricus enzyme (ssIGPS) at high temperatures. The β1α1 loop is especially important in the dehydration step as it houses the general acid Lys53. We propose that loop dynamics are governed by competing interactions on the N-and C-terminal sides of the loop. We had previously shown that disrupting interactions with the N-terminal side of the loop through the N90A substitution decreases catalytic efficiency, slows down the dehydration step and quenches loop dynamics on the picosecond to millisecond timescales. Here, we show that disrupting interactions on the C-terminal side of the loop through the R64A/D65A substitutions likewise decreases catalytic efficiency, slows down the dehydration step and quenches loop dynamics. Interestingly, the triple substitution R64A/D65A/N90A leads to new µs-ms timescale loop dynamics and makes the ring-closure step rate-determining once again. These results are consistent with a model in which the β1α1 loop is maintained in a structurally dynamic state by these competing interactions, which is important for the dehydration step of catalysis. Competing interactions in other enzymes may likewise keep their loops and other structural elements appropriately mobile.

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Section: Severing Interactions With the C-terminal Side Of The β1α1 Lmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Controlling Active Site Loop Dynamics in the (β/α)8 Barrel Enzyme Indole-3-Glycerol Phosphate Synthase

2016

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

Cited by 8 publications

References 23 publications

Controlling Active Site Loop Dynamics in the (β/α)8 Barrel Enzyme Indole-3-Glycerol Phosphate Synthase

Controlling Active Site Loop Dynamics in the (β/α)8 Barrel Enzyme Indole-3-Glycerol Phosphate Synthase

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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