Determinants of Catalytic Power and Ligand Binding in Glutamate Racemase

Spies, Maria; Reese, Joseph G.; Dodd, Dylan; Pankow, Katherine L.; Blanke, Steven R.; Baudry, Jérôme

doi:10.1021/ja809660g

Cited by 31 publications

(83 citation statements)

References 83 publications

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“…GR from B . subtilis , K i = 2.7 mM) [23]. On the contrary, 1-H-benzimidazole-2-sulfonic acid and 4-hydroxybenzene-1,3-disulfonate, which are considered potent inhibitors of GR from B .…”

Section: Discussionmentioning

confidence: 99%

“…In fact, several different drugs have been reported as GR inhibitors, such as pyrazolopyrimidinediones [13], pyridodiazepine amines [17], 8-benzyl pteridine-6,7-diones [22], dipicolinate and benzoat-3-sulfonate [23], (2R,4S)-4-substituted D-glutamate analogs [18], 1-H-benzimidazole-2-sulfonic acid [24], 2,6 pyridinedicarboxylic acid [23, 25] and 4-hydroxybenzene-1,3-disulfonate [26]. …”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Biochemical Characterization of Glutamate Racemase—A New Candidate Drug Target against Burkholderia cenocepacia Infections

Israyilova¹,

Buroni²,

Forneris³

et al. 2016

PLoS ONE

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The greatest obstacle for the treatment of cystic fibrosis patients infected with the Burkholderia species is their intrinsic antibiotic resistance. For this reason, there is a need to develop new effective compounds. Glutamate racemase, an essential enzyme for the biosynthesis of the bacterial cell wall, is an excellent candidate target for the design of new antibacterial drugs. To this aim, we recombinantly produced and characterized glutamate racemase from Burkholderia cenocepacia J2315. From the screening of an in-house library of compounds, two Zn (II) and Mn (III) 1,3,5-triazapentadienate complexes were found to efficiently inhibit the glutamate racemase activity with IC50 values of 35.3 and 10.0 μM, respectively. Using multiple biochemical approaches, the metal complexes have been shown to affect the enzyme activity by binding to the enzyme-substrate complex and promoting the formation of an inhibited dimeric form of the enzyme. Our results corroborate the value of glutamate racemase as a good target for the development of novel inhibitors against Burkholderia.

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“…GR from B . subtilis , K i = 2.7 mM) [23]. On the contrary, 1-H-benzimidazole-2-sulfonic acid and 4-hydroxybenzene-1,3-disulfonate, which are considered potent inhibitors of GR from B .…”

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Biochemical Characterization of Glutamate Racemase—A New Candidate Drug Target against Burkholderia cenocepacia Infections

Israyilova¹,

Buroni²,

Forneris³

et al. 2016

PLoS ONE

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“…This leaves us with the question why glutamate should be toxic for the cell when it is the most abundant metabolite anyway. The enzyme glutamate racemase (encoded by the essential gene racE in B. subtilis) catalyzes the conversion of L-glutamate to D-glutamate that is a building block for peptidoglycan biosynthesis (30,52). Indeed, the accumulation D-glutamate was shown to be toxic for B. subtilis (30).…”

Section: Discussionmentioning

confidence: 99%

A High-Frequency Mutation in Bacillus subtilis: Requirements for the Decryptification of the gudB Glutamate Dehydrogenase Gene

Gunka¹,

Tholen²,

Gerwig³

et al. 2011

Journal of Bacteriology

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Common laboratory strains of Bacillus subtilis encode two glutamate dehydrogenases: the enzymatically active protein RocG and the cryptic enzyme GudB that is inactive due to a duplication of three amino acids in its active center. The inactivation of the rocG gene results in poor growth of the bacteria on complex media due to the accumulation of toxic intermediates. Therefore, rocG mutants readily acquire suppressor mutations that decryptify the gudB gene. This decryptification occurs by a precise deletion of one part of the 9-bp direct repeat that causes the amino acid duplication. This mutation occurs at the extremely high frequency of 10 ؊4 . Mutations affecting the integrity of the direct repeat result in a strong reduction of the mutation frequency; however, the actual sequence of the repeat is not essential. The mutation frequency of gudB was not affected by the position of the gene on the chromosome. When the direct repeat was placed in the completely different context of an artificial promoter, the precise deletion of one part of the repeat was also observed, but the mutation frequency was reduced by 3 orders of magnitude. Thus, transcription of the gudB gene seems to be essential for the high frequency of the appearance of the gudB1 mutation. This idea is supported by the finding that the transcription-repair coupling factor Mfd is required for the decryptification of gudB. The Mfd-mediated coupling of transcription to mutagenesis might be a built-in precaution that facilitates the accumulation of mutations preferentially in transcribed genes.A s the central amino group donor for nearly all biosynthetic pathways in any living cell, glutamate plays a key role in the biochemistry and physiology of all organisms (15). Investigations with Escherichia coli demonstrate that glutamate is by far the most abundant metabolite in these bacteria, accounting for ca. 40% of the internal metabolite pool (60). Moreover, glutamate is one of the most highly embedded metabolites. In the Gram-positive soil bacterium Bacillus subtilis, at least 37 reactions make use of this amino acid (42).In B. subtilis, glutamate is exclusively synthesized from 2-oxoglutarate and glutamine by the activity of glutamate synthase in the absence of exogenous glutamate or other sources of glutamate. 2-Oxoglutarate is replenished in the citric acid cycle, whereas glutamine can be synthesized with ammonium as the nitrogen source and one of the two molecules of glutamate that are generated by glutamate synthase as the acceptor. Glutamate does also serve as a precursor for proline biosynthesis and, under conditions of osmotic stress, molar concentrations of proline have to be produced (28). Thus, it is not surprising that glutamate synthesis has to be a highly efficient process and, indeed, interactions between enzymes of the branch of the citric acid cycle that generates 2-oxoglutarate and glutamate synthase have been reported (39). Glutamate can also serve as source of carbon and nitrogen. Its utilization is initiated by an oxidative deamination...

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“…9,17 These residues include (using B. subtilis numbering): Asp10, Ser11, Cys74, Asn75, Thr76, Cys185, His187 and Thr186; where Cys74 and Cys185 act as the general acid/base for racemization. Asn75 is in a unique position, forming the back “wall” of the active site directly between the catalytic cysteines—a central location for forming strong hydrogen bonds with the Cα-carboxylate of d -glutamate, as well as contributing to active site volume.…”

Section: Introductionmentioning

confidence: 99%

“…9 It is not surprising that the water-mediated contacts in GR are highly ligand dependent. A number of recent studies in other enzymes have indicated that water networks and interstitial water structure greatly depend on the particular nature of the enzyme-ligand contacts.…”

Section: Introductionmentioning

confidence: 99%

Flooding Enzymes: Quantifying the Contributions of Interstitial Water and Cavity Shape to Ligand Binding Using Extended Linear Response Free Energy Calculations

Whalen

Spies²

2013

J. Chem. Inf. Model.

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Glutamate racemase (GR) is a cofactor independent amino acid racemase that has recently garnered increasing attention as an antimicrobial drug target. There are numerous high resolution crystal structures of GR, yet these are invariably bound to either d-glutamate or very weakly bound oxygen-based salts. Recent in silico screens have identified a number of new competitive inhibitor scaffolds, which are not based on d-Glu, but exploit many of the same hydrogen bond donor positions. In silico studies on 1-H-benzimidazole-2-sulfonic acid (BISA) show that the sulfonic acid points to the back of the GR active site, in the most buried region, analogous to the C2-carboxylate binding position in the GR-d-glutamate complex. Furthermore, BISA has been shown to be the strongest nonamino acid competitive inhibitor. Previously published computational studies have suggested that a portion of this binding strength is derived from complexation with a more closed active site, relative to weaker ligands, and in which the internal water network is more isolated from the bulk solvent. In order to validate key contacts between the buried sulfonate moiety of BISA and moieties in the back of the enzyme active site, as well as to probe the energetic importance of the potentially large number of interstitial waters contacted by the BISA scaffold, we have designed several mutants of Asn75. GR-N75A removes a key hydrogen bond donor to the sulfonate of BISA, but also serves to introduce an additional interstitial water, due to the newly created space of the mutation. GR- N75L should also show the loss of a hydrogen bond donor to the sulfonate of BISA, but does not (a priori) seem to permit an additional interstitial water contact. In order to investigate the dynamics, structure, and energies of this water-mediated complexation, we have employed the extended linear response (ELR) approach for the calculation of binding free energies to GR, using the YASARA2 knowledge based force field on a set of ten GR complexes, and yielding an R-squared value of 0.85 and a RMSE of 2.0 kJ/mol. Surprisingly, the inhibitor set produces a uniformly large interstitial water contribution to the electrostatic interaction energy (⟨Vel⟩), ranging from 30 to >50%, except for the natural substrate (d-glutamate), which has only a 7% contribution of ⟨Vel⟩ from water. The broader implications for predicting and exploiting significant interstitial water contacts in ligand–enzyme complexation are discussed.

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Determinants of Catalytic Power and Ligand Binding in Glutamate Racemase

Cited by 31 publications

References 83 publications

Biochemical Characterization of Glutamate Racemase—A New Candidate Drug Target against Burkholderia cenocepacia Infections

Biochemical Characterization of Glutamate Racemase—A New Candidate Drug Target against Burkholderia cenocepacia Infections

A High-Frequency Mutation in Bacillus subtilis: Requirements for the Decryptification of the gudB Glutamate Dehydrogenase Gene

Flooding Enzymes: Quantifying the Contributions of Interstitial Water and Cavity Shape to Ligand Binding Using Extended Linear Response Free Energy Calculations

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