Structure of Chorismate Mutase-like Domain of DAHPS from Bacillus subtilis Complexed with Novel Inhibitor Reveals Conformational Plasticity of Active Site

Pratap, Shivendra; Dev, Aditya; Kumar, Vijay; Yadav, Ravi; Narwal, M.; Tomar, Shailly; Kumar, Pravindra

doi:10.1038/s41598-017-06578-1

Cited by 12 publications

(7 citation statements)

References 49 publications

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“…The role of CM as a regulatory domain mediating the inhibition by prephenate, the product of the CM reaction, on DAH7PS has been clearly described for GspDAH7PS (3,29). To determine whether prephenate has a similar inhibitory effect on PniDAH7PS, the DAH7PS catalytic activity of PniDAH7PS was examined in the presence of prephenate.…”

Section: Prephenate Inhibits Both Dah7ps and CM Activitiesmentioning

confidence: 99%

Domain cross-talk within a bifunctional enzyme provides catalytic and allosteric functionality in the biosynthesis of aromatic amino acids

Bai

Lang

Nazmi

et al. 2019

Journal of Biological Chemistry

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Edited by Ruma Banerjee Because of their special organization, multifunctional enzymes play crucial roles in improving the performance of metabolic pathways. For example, the bacterium Prevotella nigrescens contains a distinctive bifunctional protein comprising a 3-deoxy-D-arabino heptulosonate-7-phosphate synthase (DAH7PS), catalyzing the first reaction of the biosynthetic pathway of aromatic amino acids, and a chorismate mutase (CM), functioning at a branch of this pathway leading to the synthesis of tyrosine and phenylalanine. In this study, we characterized this P. nigrescens enzyme and found that its two catalytic activities exhibit substantial hetero-interdependence and that the separation of its two distinct catalytic domains results in a dramatic loss of both DAH7PS and CM activities. The protein displayed a unique dimeric assembly, with dimerization solely via the CM domain. Small angle X-ray scattering (SAXS)-based structural analysis of this protein indicated a DAH7PS-CM hetero-interaction between the DAH7PS and CM domains, unlike the homo-association between DAH7PS domains normally observed for other DAH7PS proteins. This hetero-interaction provides a structural basis for the functional interdependence between the two domains observed here. Moreover, we observed that DAH7PS is allosterically inhibited by prephenate, the product of the CM-catalyzed reaction. This allostery was accompanied by a striking conformational change as observed by SAXS, implying that altering the hetero-domain interaction underpins the allosteric inhibition. We conclude that for this C-terminal CM-linked DAH7PS, catalytic function and allosteric regulation appear to be delivered by a common mechanism, revealing a distinct and efficient evolutionary strategy to utilize the functional advantages of a bifunctional enzyme. Multifunctional enzymes, bearing two or more catalytic activities, provide exceptional contributions to the efficient and

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Section: Prephenate Inhibits Both Dah7ps and CM Activitiesmentioning

confidence: 99%

Domain cross-talk within a bifunctional enzyme provides catalytic and allosteric functionality in the biosynthesis of aromatic amino acids

Bai

Lang

Nazmi

et al. 2019

Journal of Biological Chemistry

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“…Structure-based virtual screening of various small-molecule databases and a molecular docking study revealed that CGA showed the highest docking score with PaDHQS. Previously, our group reported the inhibitory activity of CGA against chorismate mutase-like domain type 2 (CM2) of DAHP synthase and its structure in complex with CGA (8). In this study, the binding mode of CGA in the active site of PaDHQS has been investigated.…”

Section: Resultsmentioning

confidence: 99%

“…Our previous work demonstrated that CGA exhibited pathogenic growth inhibition by binding with the CM2-like regulatory domain of DAHP synthase from B. subtilis (8). However, the structural organization and the regulatory mechanism of DAHP synthase vary among bacteria.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, it was hypothesized that CGA exhibits strong antimicrobial activities by targeting some intracellular processes in bacteria, in addition to the irreversible membrane permeabilization. Previously, our group has reported inhibitory properties of CGA and binding to the regulatory chorismate mutase-2-like (CM2) domain of the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS), the first enzyme of the shikimate pathway (8).…”

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confidence: 99%

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Structural and Biochemical Analyses Reveal that Chlorogenic Acid Inhibits the Shikimate Pathway

et al. 2020

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Chlorogenic acid (CGA) is a phenolic compound with well-known antibacterial properties against pathogens. In this study, structural and biochemical characterization display the inhibitory role of CGA against the enzyme of the shikimate pathway, a well characterized drug target in several pathogens. Here, we report the crystal structures of dehydroquinate synthase (DHQS), the second enzyme of the shikimate pathway, from Providencia alcalifaciens (PaDHQS), in binary complex with NAD and ternary complex with NAD and CGA. Structural analyses reveal that CGA occupies the substrate position in the active site of PaDHQS, which disables domain movements, leaving the enzyme in open and catalysis incompetent state. The binding analyses by ITC and SPR show that CGA binds to PaDHQS with a KD value of 6.3 μM and 0.5 μM respectively. In vitro enzyme inhibition studies show that CGA inhibits PaDHQS with a Ki of 235 ± 21 μM; while it inhibits the growth of Providencia alcalifaciens, Moraxella catarrhalis, Staphylococcus aureus and Escherichia coli with MIC values of 60 to 100 μM. In the presence of aromatic amino acids supplied externally, CGA doesn't show the toxic effect. These results, along with the observations of the inhibition of DAHP regulatory domain by CGA in our previous study, suggest that CGA binds to shikimate pathway enzymes with high affinity and inhibits their catalysis, and can be further exploited for designing novel drug-like molecules. Importance The shikimate pathway is an attractive target for the development of herbicides and antimicrobial agents as it is essential in plants, bacteria and apicomplexan parasites, but absent in humans. The enzymes of shikimate pathway are conserved among bacteria. Thus, the inhibitors of the shikimate pathway will act on wide range of pathogens. We have identified that chlorogenic acid targets the enzymes of shikimate pathway. The crystal structure of dehydroquinate synthase the second enzyme of the pathway, in complex with chlorogenic acid and enzymatic inhibition studies explain the mechanism of inhibition of chlorogenic acid. These results suggest that chlorogenic acid has a good chemical scaffold and have important implications for the further development as potent inhibitor of shikimate pathway enzymes.

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“…The recently discovered regulation domains include chorismate mutase and ferredoxin-like domains (Table 1). Among these domains, the most common is chorismate mutase located in the N terminus, such as the DAHPSs from Bacillus subtilis and Listeria monocytogenes (Light et al 2012; Pratap et al 2017). The feedback inhibition of various type I β DAHPSs is more complicated than that of type I α DAHPS.…”

Section: Introductionmentioning

confidence: 99%

Isolation and biochemical characterization of a metagenome-derived 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase gene from subtropical marine mangrove wetland sediments

Zhao

Gao

et al. 2019

AMB Expr

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3-Deoxy- d -arabino-heptulosonate-7-phosphate synthase (DAHPS) is a key rate-limiting enzyme in aromatic amino acid anabolism. A new I β -type DAHPS gene ( aro1A ) was identified in a metagenomic library from subtropical marine mangrove sediment. The gene encoded a polypeptide composed of 272 amino acids and had a maximum similarity of 52.4% to a known DAHPS at the amino acid level. Multiple sequence alignment, homologous modeling, and molecular docking showed that Aro1A had the typical (β/α) 8 barrel-shaped catalytic structural domain of DAHPS. The motifs and amino acid residues involved in the combination of substrates and metal ligand were highly conservative with the known DAHPS. The putative DAHPS gene was subcloned into a pET-30a(+) vector and was overexpressed in Escherichia coli Rosetta (DE3) cells. The recombinant protein was purified to homogeneity. The maximum activity for the recombinant Aro1A protein occurred at pH 8.0 and 40 °C. Ba 2+ and Ca 2+ stimulated the activity of Aro1A protein. The enzyme showed high affinity and catalytic efficiency ( K m PEP = 19.58 μM, V max PEP = 29.02 μM min −1 , and k cat PEP / K m PEP = 0.88 s −1 μM −1 ) under optimal reaction conditions. The enzymatic property of Aro1A indicates its potential in aromatic amino acid industrial production. Electronic supplementary material The online version of this article (10.1186/s13568-019-0742-4) contains supplementary material, which is available to authorized users.

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Structure of Chorismate Mutase-like Domain of DAHPS from Bacillus subtilis Complexed with Novel Inhibitor Reveals Conformational Plasticity of Active Site

Cited by 12 publications

References 49 publications

Domain cross-talk within a bifunctional enzyme provides catalytic and allosteric functionality in the biosynthesis of aromatic amino acids

Domain cross-talk within a bifunctional enzyme provides catalytic and allosteric functionality in the biosynthesis of aromatic amino acids

Structural and Biochemical Analyses Reveal that Chlorogenic Acid Inhibits the Shikimate Pathway

Isolation and biochemical characterization of a metagenome-derived 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase gene from subtropical marine mangrove wetland sediments

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