Biochemistry and Pharmacology - Research Publications
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ItemStructural and Dynamic Requirements for Optimal Activity of the Essential Bacterial Enzyme Dihydrodipicolinate Synthase(PUBLIC LIBRARY SCIENCE, 2012-06)Dihydrodipicolinate synthase (DHDPS) is an essential enzyme involved in the lysine biosynthesis pathway. DHDPS from E. coli is a homotetramer consisting of a 'dimer of dimers', with the catalytic residues found at the tight-dimer interface. Crystallographic and biophysical evidence suggest that the dimers associate to stabilise the active site configuration, and mutation of a central dimer-dimer interface residue destabilises the tetramer, thus increasing the flexibility and reducing catalytic efficiency and substrate specificity. This has led to the hypothesis that the tetramer evolved to optimise the dynamics within the tight-dimer. In order to gain insights into DHDPS flexibility and its relationship to quaternary structure and function, we performed comparative Molecular Dynamics simulation studies of native tetrameric and dimeric forms of DHDPS from E. coli and also the native dimeric form from methicillin-resistant Staphylococcus aureus (MRSA). These reveal a striking contrast between the dynamics of tetrameric and dimeric forms. Whereas the E. coli DHDPS tetramer is relatively rigid, both the E. coli and MRSA DHDPS dimers display high flexibility, resulting in monomer reorientation within the dimer and increased flexibility at the tight-dimer interface. The mutant E. coli DHDPS dimer exhibits disorder within its active site with deformation of critical catalytic residues and removal of key hydrogen bonds that render it inactive, whereas the similarly flexible MRSA DHDPS dimer maintains its catalytic geometry and is thus fully functional. Our data support the hypothesis that in both bacterial species optimal activity is achieved by fine tuning protein dynamics in different ways: E. coli DHDPS buttresses together two dimers, whereas MRSA dampens the motion using an extended tight-dimer interface.
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ItemCharacterisation of the First Enzymes Committed to Lysine Biosynthesis in Arabidopsis thaliana(PUBLIC LIBRARY SCIENCE, 2012-07-05)In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR) catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from Arabidopsis thaliana. The A. thaliana DHDPS enzyme (At-DHDPS2) has similar activity to the bacterial form of the enzyme, but is more strongly allosterically inhibited by (S)-lysine. Structural studies of At-DHDPS2 show (S)-lysine bound at a cleft between two monomers, highlighting the allosteric site; however, unlike previous studies, binding is not accompanied by conformational changes, suggesting that binding may cause changes in protein dynamics rather than large conformation changes. DHDPR from A. thaliana (At-DHDPR2) has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported. While all known bacterial DHDPR enzymes have a tetrameric structure, analytical ultracentrifugation, and scattering data unequivocally show that At-DHDPR2 exists as a dimer in solution. The exact arrangement of the dimeric protein is as yet unknown, but ab initio modelling of x-ray scattering data is consistent with an elongated structure in solution, which does not correspond to any of the possible dimeric pairings observed in the X-ray crystal structure of DHDPR from other organisms. This increased knowledge of the structure and function of plant lysine biosynthetic enzymes will aid future work aimed at improving primary production.
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ItemNo Preview AvailableAr-DHDPS2( 2011)
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ItemNo Preview AvailableThe crystal structures of native and (S)-lysine-bound dihydrodipicolinate synthase from Escherichia coli with improved resolution show new features of biological significance( 2008)Dihydrodipicolinate synthase (DHDPS) mediates the key first reaction common to the biosynthesis of (S)-lysine and mesodiaminopimelate. The activity of DHDPS is allosterically regulated by the feedback inhibitor (S)-lysine. The crystal structure of DHDPS from Escherichia coli has previously been published, but to only a resolution of 2.5 A, and the structure of the lysine-bound adduct was known to only 2.94 A resolution. Here, the crystal structures of native and (S)- lysine-bound dihydrodipicolinate synthase from E. coli are presented to 1.9 and 2.0 A, respectively, resolutions that allow, in particular, more accurate definition of the protein structure. The general architecture of the active site is found to be consistent with previously determined structures, but with some important differences. Arg138, which is situated at the entrance of the active site and is thought to be involved in substrate binding, has an altered conformation and is connected via a water molecule to Tyr133 of the active-site catalytic triad. This suggests a hitherto unknown function for Arg138 in the DHDPS mechanism. Additionally, a reevaluation of the dimer-dimer interface reveals a more extensive network of interactions than first thought. Of particular interest is the higher resolution structure of DHDPS with (S)-lysine bound at the allosteric site, which is remote to the active site, although connected to it by a chain of conserved water molecules. (S)-Lysine has a slightly altered conformation from that originally determined and does not appear to alter the DHDPS structure as others have reported.