Molecular evolution of an oligomeric biocatalyst functioning in lysine biosynthesis

Abstract

Dihydrodipicolinate synthase (DHDPS) is critical to the production of lysine through the diaminopimelate (DAP) pathway. Elucidation of the function, regulation and structure of this key class I aldolase has been the focus of considerable study in recent years, given that the dapA gene encoding DHDPS has been found to be essential to bacteria and plants. Allosteric inhibition by lysine is observed for DHDPS from plants and some bacterial species, the latter requiring a histidine or glutamate at position 56 (Escherichia coli numbering) over a basic amino acid. Structurally, two DHDPS monomers form the active site, which binds pyruvate and (S)-aspartate β-semialdehyde, with most dimers further dimerising to form a tetrameric arrangement around a solvent-filled centre cavity. 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 DHDPS enzymes. Finally, the potential role of pyruvate in providing substrate-mediated stabilisation of DHDPS is considered.

Keywords: Allostery; Antibiotic; Crystal; Herbicide; SAXS; Sedimentation.

Fig. 1

Diaminopimelate (DAP) biosynthesis pathway of bacteria and plants. The pathway commences with the condensation of pyruvate with (S)-aspartate β-semialdehyde [(S)-ASA] to form the heterocyclic product, (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid (HTPA) catalysed by dihydrodipicolinate synthase (DHDPS). HTPA is subsequently reduced by dihydrodipicolinate reductase (DHDPR) to form 2,3,4,5-tetrahydrodipicolinate (THDP), which is then converted via one of four sub-pathways depending on the species to meso-2,6-DAP (meso-DAP). Lysine is then formed by the decarboxylation of meso-DAP by diaminopimelate decarboxylase (DAPDC). The pathway is regulated by feedback inhibition by lysine, which binds allosterically to DHDPS

Fig. 2

Schematic of the DHDPS-catalysed reaction. Shown is the condensation of pyruvate and (S)-ASA to form HTPA and water catalysed by DHDPS (EC 4.3.3.7)

Fig. 3

Structure of (a) active site and (b) allosteric site of DHDPS. a The active site of pyruvate-bound Vitis vinifera DHDPS [Protein Data Bank (PDB) ID: 3TUU] with labelled residues annotated according to E. coli DHDPS numbering. b The allosteric binding cleft of V. vinifera DHDPS co-crystallised with lysine (PDB ID: 4HNN). Labelled are the two bound lysine ligands that mediate allosteric inhibition of the enzyme

Fig. 4

Diverse quaternary structures of bacterial and plant DHDPS enzymes. a Bacterial ‘head-to-head’ DHDPS tetramer (PDB ID: 3HIJ), b Staphylococcus aureus DHDPS dimer (PDB ID: 3DAQ) and c plant ‘back-to-back’ DHDPS tetramer (PDB ID: 3TUU)