Complex Formation between Two Biosynthetic Enzymes Modifies the Allosteric Regulatory Properties of Both

Blackmore, Nicola J.; Nazmi, Ali Reza; Hutton, Richard D.; Webby, Melissa N.; Baker, Edward N.; Jameson, Geoffrey B.; Parker, Emily J.

doi:10.1074/jbc.m115.638700

Cited by 29 publications

(59 citation statements)

References 34 publications

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“…Sedimentation velocity experiments were carried out at three protein concentrations under the conditions previously described for the wild type enzyme [21]. At the lowest analyzed concentration, 0.09 mg/mL, a major species with a sedimentation coefficient of 4.0 S can be observed (Fig 4A).…”

Section: Resultsmentioning

confidence: 99%

Quaternary structure is an essential component that contributes to the sophisticated allosteric regulation mechanism in a key enzyme from Mycobacterium tuberculosis

et al. 2017

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The first enzyme of the shikimate pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS), adopts a range of distinct allosteric regulation mechanisms in different organisms, related to different quaternary assemblies. DAH7PS from Mycobacterium tuberculosis (MtuDAH7PS) is a homotetramer, with the allosteric sites in close proximity to the interfaces. Here we examine the importance of the quaternary structure on catalysis and regulation, by amino acid substitution targeting the tetramer interface of MtuDAH7PS. Using only single amino acid substitutions either in, or remote from the interface, two dimeric variants of MtuDAH7PS (MtuDAH7PSF227D and MtuDAH7PSG232P) were successfully generated. Both dimeric variants maintained activity due to the distance between the sites of amino acid substitution and the active sites, but attenuated catalytic efficiency was observed. Both dimeric variants showed significantly disrupted allosteric regulation with greatly impaired binding affinity for one of the allosteric ligands. Molecular dynamics simulations revealed changes in protein dynamics and average conformations in the dimeric variant caused by amino acid substitution remote to the tetramer interface (MtuDAH7PSG232P), which are consistent with the observed reduction in catalytic efficiency and loss of allosteric response.

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Section: Resultsmentioning

confidence: 99%

Quaternary structure is an essential component that contributes to the sophisticated allosteric regulation mechanism in a key enzyme from Mycobacterium tuberculosis

et al. 2017

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“…Furthermore, MtuDAH7PS G190P retained the ability to activate MtuCM, demonstrating that the inter-enzyme interaction was intact ( Table 1). [16][17][18] Both the wildtype enzyme and substituted MtuDAH7PS enhanced the activity of MtuCM over 100-fold in similar ways, by lowering the KM value for chorismate and enhancing turnover.…”

Section: Sequence Analysis and Protein Sectors In Type II Dah7psmentioning

confidence: 93%

“…It has been shown that the allosteric machinery of MtuDAH7PS can be utilized by another small protein partner, chorismate mutase (MtuCM), by forming a noncovalent complex. [16][17][18] CM catalyzes a downstream reaction in the shikimate pathway, converting chorismate to prephenate, and is at the start of the branchpoint of the shikimate pathway that leads to the production of Phe and Tyr. It has been shown that complex formation between MtuDAH7PS and MtuCM significantly enhances the catalytic activity of MtuCM.…”

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“…It has been shown that complex formation between MtuDAH7PS and MtuCM significantly enhances the catalytic activity of MtuCM. [16][17][18] Furthermore, the normally unregulated MtuCM becomes sensitive to inhibition by Phe, which binds to the dimer interface on MtuDAH7PS. [16][17][18] A similar scenario was also observed in another type II enzyme, i.e.…”

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“…[16][17][18] Furthermore, the normally unregulated MtuCM becomes sensitive to inhibition by Phe, which binds to the dimer interface on MtuDAH7PS. [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.…”

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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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Biosynthesis of Aromatic Amino Acids and Its Regulation

Cohen¹

2016

Microbial Biochemistry

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Glucose is an aliphatic substance, and so the synthesis of phenylalanine, tyrosine and tryptophan from it, presents the biochemist with the problem of the biosynthesis of the aromatic nucleus.The key to this problem was given 50 years ago by the isolation of polyauxotrophic mutants which required the presence of the three aromatic amino acids in the medium for growth, and in addition p-aminobenzoic acid (a precursor of folic acid). The existence of this multiple requirement as a result of a single mutation immediately suggested that the genetic block consisted in the inability to synthesize a precursor common to all four essential metabolites. After trying 55 cyclic and hydroaromatic derivatives without success, B. D. Davis discovered that shikimic acid could replace the four growth factors. Moreover, growth was proportional to the amount of shikimate added. Other bacterial mutants, in which the lesion lies at a later step in aromatic biosynthesis, accumulate shikimic acid in the medium. It was therefore concluded that shikimic acid was the precursor sought; however, its identification relied only on chromatographic analysis and on the response given by mutants. Later, the compound was isolated and characterized by the classical methods of organic chemistry.Shikimic acid has been known since the nineteenth century as a rare constituent of certain plants. We shall now examine how this compound is formed in E. coli and how it leads to chorismic acid, from which the different aromatic compounds diverge.

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Complex Formation between Two Biosynthetic Enzymes Modifies the Allosteric Regulatory Properties of Both

Cited by 29 publications

References 34 publications

Quaternary structure is an essential component that contributes to the sophisticated allosteric regulation mechanism in a key enzyme from Mycobacterium tuberculosis

Quaternary structure is an essential component that contributes to the sophisticated allosteric regulation mechanism in a key enzyme from Mycobacterium tuberculosis

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

Biosynthesis of Aromatic Amino Acids and Its Regulation

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