Quaternary Structure Is an Essential Component That Contributes to the Sophisticated Allosteric Regulation Mechanism in a Key Enzyme From Mycobacterium Tuberculosis

PLoS One. 2017 Jun 30;12(6):e0180052. doi: 10.1371/journal.pone.0180052. eCollection 2017.

Abstract

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.

MeSH terms

  • 3-Deoxy-7-Phosphoheptulonate Synthase / chemistry*
  • 3-Deoxy-7-Phosphoheptulonate Synthase / genetics
  • 3-Deoxy-7-Phosphoheptulonate Synthase / metabolism
  • Allosteric Regulation
  • Amino Acid Substitution
  • Crystallography, X-Ray
  • Dimerization
  • Molecular Dynamics Simulation
  • Mutagenesis
  • Mycobacterium tuberculosis / enzymology*
  • Phenylalanine / metabolism
  • Protein Structure, Quaternary
  • Tryptophan / metabolism

Substances

  • Phenylalanine
  • Tryptophan
  • 3-Deoxy-7-Phosphoheptulonate Synthase

Grant support

This work was supported by the Maurice Wilkins Centre for Molecular Biodiscovery New Zealand Marsden fund (UOC1105) and the Biomolecular Interaction Centre. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.