Structural studies of saccharopine reductase and purple acid phosphatase

Abstract

Lysine is synthesised in two different ways in living organisms, either by the diaminopimelate pathway or the [alpha]-aminoadipate pathway. These routes belong to the aspartate and the glutamate family of amino acid biosynthesis, respectively. Bacteria, plants and lower fungi make use of the diaminopimelate pathway whereas higher fungi and euglenoid algae use the [alpha]-aminoadipate pathway. Saccharopine reductase (EC 1.5.1.10) is an NADPH-dependent enzyme catalysing the penultimate step of the [alpha]-aminoadipate pathway where the lysine precursor saccharopine is formed from glutamate and [alpha]-aminoadipate-[delta]-semialdehyde. The three-dimensional structure of this enzyme from die rice blast fungus, Magnaporthe grisea, has been determined by x-ray crystallography. The structure of the apo enzyme was determined by multiple wavelength anomalous diffraction and refined at 2.0 A resolution. The structures of three complexes of saccharopine reductase have also been determined: the ternary complex with saccharopine and NADPH and the binary complexes with NADPH or the potential substrate analogue cyclohexanecarboxylate, respectively.

These crystal structures have facilitated a detailed characterisation of the enzyme's active site and a reaction mechanism is suggested where the enzyme places the reactants at favourable distances for the reaction to take place. Large domain movements in the enzyme seem to occur during the course of the reaction. Even though saccharopine reductase is part of the glutamate family of amino acid biosynthesis, the enzyme is found to be similar to enzymes in the aspartate family. This suggests an evolutionary relationship between the two families.

Purple acid phosphatases are enzymes containing a binuclear metal-centre M(II)-Fe(III), where M is Zn or Mn for plant enzymes and Fe for mammalian enzymes. The enzyme's name originates from its intense purple colour, which is due to a tyrosine-to-iron charge-transfer transition. The exact physiological function of purple acid phosphatases is unknown. However, the mammalian bone enzyme has been suggested to be involved in bone resorption. The structure of purple acid phosphatase from rat bone (EC 3.1.3.2) has been determined at 2.2 A resolution using the molecular replacement method. The core of the mammalian enzyme is found to be structurally very similar to the plant enzyme, although differences are found in the architecture of the active site cleft. Furthermore, the structure provides evidence for a [my]-(hydr)oxo bridge in the binuclear iron-centre of the protein. A mechanism is suggested based on the structure, where the phosphate group of the substrate is co-ordinated to the Fe(II) ion and a hydroxyl group, bound to the Fe(III) ion, performs a nucleophilic attack on the phosphoester-bond that is cleaved.