Structural analysis of substrate-mimicking inhibitors in complex with Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase – The importance of accommodating the active site water

Heyes, L.C.; Reichau, Sebastian; Cross, Penelope J.; Jameson, Geoffrey B.; Parker, Emily J.

doi:10.1016/j.bioorg.2014.08.003

Cited by 7 publications

(7 citation statements)

References 40 publications

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“…Hydrogen bonds between Lys107 and residues Glu176, His209, and the backbone carbonyl of His210 of the opposing chain are lost, whereas new hydrogen bonds are established between Lys107 and Glu98 and Gln172 of the opposing chain. Likewise, hydrogen bonds between Glu98 and residues Thr151, Tyr155 and the backbone of Asp148 are lost, as is the hydrogen bond that links the backbone carbonyl of the catalytic Lys99 with Gln172 of the opposing chain. The β 2 α 2 loop contributes several residues of importance to substrate binding and catalysis and the local changes in conformation observed in this structure relative to the Phe-free protein are likely to be associated with the changes in catalytic properties observed for this protein in the presence of Phe: a 4-fold reduction in k cat and 2-fold increases of the apparent K m values for both substrates (Figure S2).…”

Section: Resultsmentioning

confidence: 99%

“…In the apo form, repulsive Coulombic interactions exist between catalytically important residues Lys188 and Lys99, both of which are involved in the protontransfer mechanisms during catalysis, Arg94 which binds the carboxylate of PEP, as well as Arg236 and Arg101 which bind the phosphate of PEP and E4P respectively. 26 This network of interactions among positively charged residues, which is only present in the absence of Phe, is likely to maintain the correct relative distances between key residues, thereby maintaining the most catalytically active conformation. Additionally, Glu145 interacts more favorably with Lys99 and Arg94 in the absence of Phe.…”

Section: Allosteric Regulation Triggers Local Flexibility Changes And...mentioning

confidence: 99%

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Calculated pK_a Variations Expose Dynamic Allosteric Communication Networks

Lang¹,

Heyes²,

Jameson

et al. 2016

J. Am. Chem. Soc.

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Allosteric regulation of protein function, the process by which binding of an effector molecule provokes a functional response from a distal site, is critical for metabolic pathways. Yet, the way the allosteric signal is communicated remains elusive, especially in dynamic, entropically driven regulation mechanisms for which no major conformational changes are observed. To identify these dynamic allosteric communication networks, we have developed an approach that monitors the pKa variations of ionizable residues over the course of molecular dynamics simulations performed in the presence and absence of an allosteric regulator. As the pKa of ionizable residues depends on their environment, it represents a simple metric to monitor changes in several complex factors induced by binding an allosteric effector. These factors include Coulombic interactions, hydrogen bonding, and solvation, as well as backbone motions and side chain fluctuations. The predictions that can be made with this method concerning the roles of ionizable residues for allosteric communication can then be easily tested experimentally by changing the working pH of the protein or performing single point mutations. To demonstrate the method's validity, we have applied this approach to the subtle dynamic regulation mechanism observed for Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, the first enzyme of aromatic biosynthesis. We were able to identify key communication pathways linking the allosteric binding site to the active site of the enzyme and to validate these findings experimentally by reestablishing the catalytic activity of allosterically inhibited enzyme via modulation of the working pH, without compromising the binding affinity of the allosteric regulator.

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Section: Resultsmentioning

confidence: 99%

Section: Allosteric Regulation Triggers Local Flexibility Changes And...mentioning

confidence: 99%

Calculated pK_a Variations Expose Dynamic Allosteric Communication Networks

Lang¹,

Heyes²,

Jameson

et al. 2016

J. Am. Chem. Soc.

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“…The K186 Nε···N oxime hydrogen bond is important for inhibitor binding, with K i increasing 180-fold in the K186A mutant, corresponding to a hydrogen bonding energy of −2.8 kcal/mol. Other factors that could contribute to the inhibitor’s high affinity include the planar geometry at C2, the two crystallographic waters that occupy the same space as PEP’s nonbridging phosphate oxygens, and inhibitor-induced protein dynamic changes …”

Section: Discussionmentioning

confidence: 99%

Linear Free Energy Relationship Analysis of Transition State Mimicry by 3-Deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) Oxime, a DAHP Synthase Inhibitor and Phosphate Mimic

Balachandran

Berti

2017

Biochemistry

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3-Deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to form DAHP in the first step of the shikimate biosynthetic pathway. DAHP oxime, in which an oxime replaces the ketone, is a potent inhibitor, with K = 1.5 μM. Linear free energy relationship (LFER) analysis of DAHP oxime inhibition using DAHP synthase mutants revealed an excellent correlation between transition state stabilization and inhibition. The equations of LFER analysis were rederived to formalize the possibility of proportional, rather than equal, changes in the free energies of transition state stabilization and inhibitor binding, in accord with the fact that the majority of LFER analyses in the literature demonstrate nonunity slopes. A slope of unity, m = 1, indicates that catalysis and inhibitor binding are equally sensitive to perturbations such as mutations or modified inhibitor/substrate structures. Slopes <1 or >1 indicate that inhibitor binding is less sensitive or more sensitive, respectively, to perturbations than is catalysis. LFER analysis using the tetramolecular specificity constant, that is, plotting log(KKK/k) versus log(K), revealed a slope, m, of 0.34, with r = 0.93. This provides evidence that DAHP oxime is mimicking the first irreversible transition state of the DAHP synthase reaction, presumably phosphate departure from the tetrahedral intermediate. This is evidence that the oxime group can act as a functional, as well as structural, mimic of phosphate groups.

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“…In this study, we combined transcriptome and targeted metabolite profile analyses to explore potential enzymes or pathways involved in the differential regulation of phthalide accumulation. Six enzymes, including phospho-2-dehydro-3deoxyheptonate aldolase 2 ( 17 eukaryotes, and plants (Bagautdinov and Kunishima, 2007;Heyes et al, 2014). Polyphenol oxidase ( 36) is a group of Cu-containing enzymes that catalyzes the oxidation of several phenols to o-quinones (Prexler et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

Investigation of Enzymes in the Phthalide Biosynthetic Pathway in Angelica sinensis Using Integrative Metabolite Profiles and Transcriptome Analysis

et al. 2022

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The roots of Angelica sinensis (Oliv.) Diels are well known for their efficacy in promoting blood circulation. Although many studies have indicated that phthalides are the main chemical components responsible for the pharmacological properties of A. sinensis, the phthalide biosynthetic pathway and enzymes that transform different phthalides are still poorly understood. We identified 108 potential candidate isoforms for phthalide accumulation using transcriptome and metabolite profile analyses. Then, six enzymes, including phospho-2-dehydro-3-deoxyheptonate aldolase 2, shikimate dehydrogenase, primary amine oxidase, polyphenol oxidase, tyrosine decarboxylase, and shikimate O-hydroxycinnamoyl transferase, were identified and proven to be involved in phthalide accumulation by heterologously expressing these proteins in Escherichia coli. We proposed a possible mechanism underlying phthalide transformation and biosynthetic pathways in A. sinensis based on our findings. The results of our study can provide valuable information for understanding the mechanisms underlying phthalide accumulation and transformation and enable further development of quality control during the cultivation of A. sinensis.

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Structural analysis of substrate-mimicking inhibitors in complex with Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase – The importance of accommodating the active site water

Cited by 7 publications

References 40 publications

Calculated pK_a Variations Expose Dynamic Allosteric Communication Networks

Calculated pK_a Variations Expose Dynamic Allosteric Communication Networks

Linear Free Energy Relationship Analysis of Transition State Mimicry by 3-Deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) Oxime, a DAHP Synthase Inhibitor and Phosphate Mimic

Investigation of Enzymes in the Phthalide Biosynthetic Pathway in Angelica sinensis Using Integrative Metabolite Profiles and Transcriptome Analysis

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Structural analysis of substrate-mimicking inhibitors in complex with Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase – The importance of accommodating the active site water

Cited by 7 publications

References 40 publications

Calculated pKa Variations Expose Dynamic Allosteric Communication Networks

Calculated pKa Variations Expose Dynamic Allosteric Communication Networks

Linear Free Energy Relationship Analysis of Transition State Mimicry by 3-Deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) Oxime, a DAHP Synthase Inhibitor and Phosphate Mimic

Investigation of Enzymes in the Phthalide Biosynthetic Pathway in Angelica sinensis Using Integrative Metabolite Profiles and Transcriptome Analysis

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Calculated pK_a Variations Expose Dynamic Allosteric Communication Networks

Calculated pK_a Variations Expose Dynamic Allosteric Communication Networks