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.
Highlights
Protein complexes with different quaternary structures are essential functional modules within protein interaction networks in the cellular environment [1]
Our results demonstrate that changing the quaternary structure of MtuDAH7PS significantly reduces its allosteric response to aromatic amino acids, and impairs catalytic function, exposing the dynamic connections between remote sites
In the ligand-free MtuDAH7PS crystal structure (PDB 3NV8), the dimer interface involves more than 50 residues from each chain and has a buried area of 1860 Å2
Summary
Protein complexes with different quaternary structures are essential functional modules within protein interaction networks in the cellular environment [1]. E24 μM MtuDAH7PSG232P and a background of 50 μM Phe present in the cell with a 5 mM Trp titrant. F28 μM MtuDAH7PSF227D and a background of 50 μM Phe present in the cell with a 5 mM Trp titrant. Quaternary structure of DAH7PS from Mycobacterium tuberculosis to bind at the preserved dimeric interface, were very similar to that for the wild-type enzyme (Kd = 21 ± 1 μM) [24]. Given that protein dynamics has previously been shown to play an important role in the allosteric regulation of MtuDAH7PS, molecular dynamics (MD) simulations were conducted for the dimeric variant MtuDAH7PSG232P, the theoretical wild-type dimer MtuDAH7PSWTdimer and the tetrameric MtuDAH7PSWT in order to examine the effects of dimerization on the dynamic properties of this enzyme. All systems were equilibrated and analysis were conducted using only trajectories from the equilibrated time period (S1 Fig)
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