Inter-Enzyme Allosteric Regulation of Chorismate Mutase in <i>Corynebacterium glutamicum</i>: Structural Basis of Feedback Activation by Trp

Burschowsky, D.; Thorbjornsrud, H.V.; Heim, Joel B.; Fahrig‐Kamarauskaitė, Jūratė; Würth-Roderer, Kathrin; Kast, Peter; Krengel, Ute

doi:10.1021/acs.biochem.7b01018

Cited by 28 publications

(45 citation statements)

References 92 publications

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“…[16][17][18] A similar scenario was also observed in another type II enzyme, i.e. the DAH7PS from Corynebacterium glutamicum, 19 indicating this inter-enzyme communication may be a more common feature among the type II DAH7PS enzymes.…”

supporting

confidence: 61%

“…Allosteric regulation of known type II DAH7PS enzymes is dynamically driven. 15,19,26 Among these, MtuDAH7PS shows the most complex dynamic allosteric response. MtuDAH7PS utilizes four distinct functional sites, including one active site and three allosteric sites that are specific to each of the three aromatic amino acids (Trp, Phe and Tyr).…”

Section: Discussionmentioning

confidence: 99%

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A single amino acid substitution uncouples catalysis and allostery in an essential biosynthetic enzyme in Mycobacterium tuberculosis

Jiao

Fan

Blackmore

et al. 2020

Journal of Biological Chemistry

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Allostery exploits the conformational dynamics of enzymes by triggering a shift in population ensembles toward functionally distinct conformational or dynamic states. Allostery extensively regulates the activities of key enzymes within biosynthetic pathways to meet metabolic demand for their end products. Here, we have examined a critical enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS), at the gateway to aromatic amino acid biosynthesis in Mycobacterium tuberculosis, which shows extremely complex dynamic allostery: three distinct aromatic amino acids jointly communicate occupancy to the active site via subtle changes in dynamics, enabling exquisite fine-tuning of delivery of these essential metabolites. Furthermore, this allosteric mechanism is co-opted by pathway branchpoint enzyme chorismate mutase upon complex formation. In this study, using statistical coupling analysis, site-directed mutagenesis, isothermal calorimetry, small-angle X-ray scattering, and X-ray crystallography analyses, we have pinpointed a critical node within the complex dynamic communication network responsible for this sophisticated allosteric machinery. Through a facile Gly to Pro substitution, we have altered backbone dynamics, completely severing the allosteric signal yet remarkably, generating a nonallosteric enzyme that retains full catalytic activity. We also identified a second residue of prime importance to the inter-enzyme communication with chorismate mutase. Our results reveal that highly complex dynamic allostery is surprisingly vulnerable and provide further insights into the intimate link between catalysis and allostery.

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supporting

confidence: 61%

Section: Discussionmentioning

confidence: 99%

A single amino acid substitution uncouples catalysis and allostery in an essential biosynthetic enzyme in Mycobacterium tuberculosis

Jiao

Fan

Blackmore

et al. 2020

Journal of Biological Chemistry

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“…Unlike in proline, this conformation is, however, not permanently fixed, allowing for greater conformational sampling. Such a temporary "kink-potential" is probably crucial for DS-dependent activity switching of AroQδsubclass CMs (1,2,11).…”

Section: Discussionmentioning

confidence: 99%

“…In this study, we probed the structural requirements and mechanisms for the activity switch of MtCM and whether or not interaction with MtDS is mandatory for efficient catalysis. We have employed diverse cycles of directed evolution to improve the mediocre efficiency of MtCM as the prototype for catalytically impaired AroQδ enzymes (1,2,11). Our results reveal mutation patterns and structural changes responsible for high activity and demonstrate that MtCM is inherently capable of efficient catalysis despite the lack of crucial active site residues, which are otherwise strongly conserved in the AroQ family.…”

Section: Introductionmentioning

confidence: 97%

“…Typically, this type of regulation employs allosteric sites on the enzyme itself. Alternatively, an allosteric site can be temporarily provided by transient protein-protein interactions, also called 'inter-enzyme allostery', as first described for shikimate pathway enzymes from Mycobacterium tuberculosis H37Rv (1) and more recently also from Corynebacterium glutamicum (2).…”

Section: Introductionmentioning

confidence: 99%

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Evolving the naturally compromised chorismate mutase from Mycobacterium tuberculosis to top performance

Fahrig-Kamarauskait≑

Würth-Roderer

Thorbjornsrud

et al. 2020

Journal of Biological Chemistry

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Chorismate mutase (CM), an essential enzyme at the branch point of the shikimate pathway, is required for the biosynthesis of phenylalanine and tyrosine in bacteria, archaea, plants, and fungi. MtCM, the CM from Mycobacterium tuberculosis, has less than 1% of the catalytic efficiency of a typical natural CM and requires complex formation with DAHP synthase for high activity. To explore the full potential of MtCM for catalyzing its native reaction, we applied diverse iterative cycles of mutagenesis and selection, thereby raising kcat/Km 270-fold to 5 × 105 M-1s-1, which is even higher than for the complex. Moreover, the evolutionarily optimized autonomous MtCM, which had 11 of its 90 amino acids exchanged, was stabilized compared to its progenitor, as indicated by a 9 °C increase in melting temperature. The 1.5 Å crystal structure of the top-evolved MtCM variant reveals the molecular underpinnings of this activity boost. Some acquired residues, (e.g. Pro52 and Asp55) are conserved in naturally efficient CMs, but most of them lie beyond the active site. Our evolutionary trajectories reached a plateau at the level of the best natural enzymes suggesting that we have exhausted the potential of MtCM. Taken together, these findings show that the scaffold of MtCM, which naturally evolved for mediocrity to enable inter-enzyme allosteric regulation of the shikimate pathway, is inherently capable of high activity.

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Role of UDP‐N‐acetylmuramic acid in the regulation of MurA activity revealed by molecular dynamics simulations

de Oliveira,

da Costa,

Silva

et al. 2024

Protein Science

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The peptidoglycan biosynthesis pathway plays a vital role in bacterial cells, and facilitates peptidoglycan layer formation, a fundamental structural component of the bacterial cell wall. The enzymes in this pathway are candidates for antibiotic development, as most do not have mammalian homologues. The UDP‐N‐acetylglucosamine (UNAG) enolpyruvyl transferase enzyme (MurA) in the peptidoglycan pathway cytoplasmic step is responsible for the phosphoenolpyruvate (PEP)–UNAG catalytic reaction, forming UNAG enolpyruvate and inorganic phosphate. Reportedly, UDP‐N‐acetylmuramic acid (UNAM) binds tightly to MurA forming a dormant UNAM–PEP–MurA complex and acting as a MurA feedback inhibitor. MurA inhibitors are complex, owing to competitive binding interactions with PEP, UNAM, and UNAG at the MurA active site. We used computational methods to explore UNAM and UNAG binding. UNAM showed stronger hydrogen‐bond interactions with the Arg120 and Arg91 residues, which help to stabilize the closed conformation of MurA, than UNAG. Binding free energy calculations using end‐point computational methods showed that UNAM has a higher binding affinity than UNAG, when PEP is attached to Cys115. The unbinding process, simulated using τ–random acceleration molecular dynamics, showed that UNAM has a longer relative residence time than UNAG, which is related to several complex dissociation pathways, each with multiple intermediate metastable states. This prevents the loop from opening and exposing the Arg120 residue to accommodate UNAG and potential new ligands. Moreover, we demonstrate the importance of Cys115‐linked PEP in closed‐state loop stabilization. We provide a basis for evaluating novel UNAM analogues as potential MurA inhibitors.Public significanceMurA is a critical enzyme involved in bacterial cell wall biosynthesis and is involved in antibiotic resistance development. UNAM can remain in the target protein's active site for an extended time compared to its natural substrate, UNAG. The prolonged interaction of this highly stable complex known as the ‘dormant complex’ comprises UNAM–PEP–MurA and offers insights into antibiotic development, providing potential options against drug‐resistant bacteria and advancing our understanding of microbial biology.

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Inter-Enzyme Allosteric Regulation of Chorismate Mutase in Corynebacterium glutamicum: Structural Basis of Feedback Activation by Trp

Cited by 28 publications

References 92 publications

A single amino acid substitution uncouples catalysis and allostery in an essential biosynthetic enzyme in Mycobacterium tuberculosis

A single amino acid substitution uncouples catalysis and allostery in an essential biosynthetic enzyme in Mycobacterium tuberculosis

Evolving the naturally compromised chorismate mutase from Mycobacterium tuberculosis to top performance

Role of UDP‐N‐acetylmuramic acid in the regulation of MurA activity revealed by molecular dynamics simulations

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