Structural, kinetic and computational investigation of Vitis vinifera DHDPS reveals new insight into the mechanism of lysine-mediated allosteric inhibition

  title={Structural, kinetic and computational investigation of Vitis vinifera DHDPS reveals new insight into the mechanism of lysine-mediated allosteric inhibition},
  author={Sarah C Atkinson and Con Dogovski and Matthew T. Downton and Peter E. Czabotar and Renwick C. J. Dobson and Juliet A Gerrard and John Wagner and Matthew A. Perugini},
  journal={Plant Molecular Biology},
Lysine is one of the most limiting amino acids in plants and its biosynthesis is carefully regulated through inhibition of the first committed step in the pathway catalyzed by dihydrodipicolinate synthase (DHDPS). This is mediated via a feedback mechanism involving the binding of lysine to the allosteric cleft of DHDPS. However, the precise allosteric mechanism is yet to be defined. We present a thorough enzyme kinetic and thermodynamic analysis of lysine inhibition of DHDPS from the common… 
Tyrosine 110 plays a critical role in regulating the allosteric inhibition of Campylobacter jejuni dihydrodipicolinate synthase by lysine.
The high-resolution crystal structures of DHDPS from Campylobacter jejuni with and without its inhibitor bound to the allosteric sites reveal a role for Y110 in the regulation of theAllosteric inhibition by lysine.
Dynamic Modelling Reveals ‘Hotspots’ on the Pathway to Enzyme-Substrate Complex Formation
A metastable binding intermediate characterized by multiple points of intermolecular interaction between pyruvate and key DHDPS residue Arg140 was found to be a highly conserved feature of the binding trajectory when comparing alternative binding pathways and these binding intermediates are thermodynamically metastable.
Differential lysine‐mediated allosteric regulation of plant dihydrodipicolinate synthase isoforms
Compared the catalytic activity, lysine-mediated inhibition and structures of both A. thaliana DHDPS isoforms, it is found that although the recombinantly produced enzymes have similar kinetic properties, AtDHDPS1 is 10-fold more sensitive to l Lysine.
Molecular evolution of an oligomeric biocatalyst functioning in lysine biosynthesis
The architecture and behaviour of these dimer-of-dimers is explored in detail, including biophysical studies utilising analytical ultracentrifugation, small-angle X-ray scattering and macromolecular crystallography that show bacterial DHDPS tetramers adopt a head-to-head quaternary structure, compared to the back- to-back arrangement observed for plant D HDPS enzymes.
Biomimetic Design Results in a Potent Allosteric Inhibitor of Dihydrodipicolinate Synthase from Campylobacter jejuni.
The first potent inhibitor of DHDPS is designed by mimicking its natural allosteric regulation by lysine, and the crystal structure of the protein-inhibitor complex at 2.2 Å resolution is obtained.
Dihydrodipicolinate synthase from Campylobacter jejuni: kinetic mechanism of cooperative allosteric inhibition and inhibitor-induced substrate cooperativity.
It has been found that ASA binds cooperatively in the presence of (S)-lysine, and the cooperativity of binding increases at near-KM concentrations of pyruvate, which differs from the kinetic models for inhibition reported for DHDPS from other sources.
Evidence of Allosteric Enzyme Regulation via Changes in Conformational Dynamics: A Hydrogen/Deuterium Exchange Investigation of Dihydrodipicolinate Synthase.
Hydrogen/deuterium exchange mass spectrometry experiments provide evidence that in CjDHDPS allostery is mediated by changes in the extent of thermally activated conformational fluctuations, illustrating why allosteric control does not have to be associated with crystallographically detectable large-scale transitions.


New insights into the mechanism of dihydrodipicolinate synthase using isothermal titration calorimetry.
Characterisation of the First Enzymes Committed to Lysine Biosynthesis in Arabidopsis thaliana
DHDPR from A. thaliana (At-D HDPR2) has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported.
Crystal structure and kinetic study of dihydrodipicolinate synthase from Mycobacterium tuberculosis.
The three-dimensional structure of the enzyme dihydrodipicolinate synthase from Mycobacterium tuberculosis (Mtb-DHDPS) was determined and refined, which shows that the (S)-lysine-binding site is not conserved in Mtb, when compared with DHDPS enzymes that are known to be inhibited by (S-lysine).
Conserved main‐chain peptide distortions: A proposed role for Ile203 in catalysis by dihydrodipicolinate synthase
Interestingly, the hydroxyl group of β‐hydroxypyruvate was hydrogen‐bonded to the main‐chain carbonyl of Ile203, which provides insight into the possible catalytic role played by this peptide unit, which has a highly strained torsion angle (ω ∼201°).
The crystal structures of native and (S)-lysine-bound dihydrodipicolinate synthase from Escherichia coli with improved resolution show new features of biological significance.
The general architecture of the active site is found to be consistent with previously determined structures, but with some important differences, and a re-evaluation of the dimer-dimer interface reveals a more extensive network of interactions than first thought.
Reaction mechanism of Escherichia coli dihydrodipicolinate synthase investigated by X-ray crystallography and NMR spectroscopy.
A reaction mechanism for DHDPS is proposed, and important features for inhibition are identified, and (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid is identified as the only product.
Structure of dihydrodipicolinate synthase of Nicotiana sylvestris reveals novel quaternary structure.
The crystal structure of Nicotiana sylvestris DHDPS with and without inhibitory lysine bound to the enzyme has been solved and a novel quaternary structure provides a structural basis for the strong inhibition of plant D HDPS enzymes by L-lysine.
Crystal, Solution and In silico Structural Studies of Dihydrodipicolinate Synthase from the Common Grapevine
This study is the first to demonstrate using both crystal and solution state measurements that DHDPS from the grapevine plant adopts an alternative tetrameric architecture to the bacterial form, which is important for optimizing protein dynamics as suggested by molecular dynamics simulations reported in this study.
Exploring the dihydrodipicolinate synthase tetramer: how resilient is the dimer-dimer interface?