Studies in (S)-lysine biosynthesis via the diaminopimelate pathway
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
The enzymes of the diaminopimelate (dap) pathway, which is responsible for the biosynthesis of (S)-lysine in plants and micro-organisms, are of interest as potential targets for herbicide and antibacterial research. This thesis has investigated two key enzymes of this pathway: dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR). DHDPS, the first enzyme committed specifically to (S)-lysine biosynthesis, catalyses the condensation of pyruvate and (S)-aspartate β-semialdehyde ((S)-ASA) to form an unstable heterocycle. Dihydrodipicolinate reductase (DHDPR), the next enzyme in the pathway, mediates the reduction of this product to tetrahydrodipicolinate. A coupled assay measuring the activities of DHDPS and DHDPR was optimised. Both DHDPS and DHDPR were purified from E. coli strains that had been genetically engineered to overproduce these enzymes. This assay monitored the utilisation of NADPH, at 340 nm, by DHDPR. Kinetic studies were performed on DHDPS, giving Km and Vmax parameters for both substrates, (S)-aspartate β-semialdehyde (synthesised from (S)-allylglycine) and pyruvate. It was shown that (S)-aspartate β-semialdehyde did not inhibit the DHDPS enzyme even at high substrate concentrations, although (S)-lysine was found to be a feedback inhibitor, acting in a reversible uncompetitive manner with respect to both substrates, thus, binding at an allosteric site. Kinetic studies were performed on DHDPR, giving Km and Vmax values for the substrate. DHDPR was shown to utilise either NADPH or NADH as a redox coenzyme. Of these NADPH is generally used in the assay due to its greater chemical stability, even though the Km for NADH is lower. Structural and enzymatic inhibition studies on DHDPS revealed that (S)-aspartate β-semialdehyde exists predominantly as a linear hydrate in solution at physiological pH. Inhibition studies on DHDPS showed that the cyclic molecule homoserine lactone and related compounds, such as 2-aminocyclopentanone, which mimic a possible cyclic form of (S)-aspartate β-semialdehyde are allosteric site inhibitors acting in a reversible noncompetitive manner. Analogues of the hydrate form of (S)-aspartate β-semialdehyde such as the amino acids (R)-cysteine sulfinic acid, and (S)-glutarnic acid inhibited in a reversible uncompetitive manner. (S)-Aspartic acid also inhibited DHDPS, but was a reversible mixed type inhibitor. NMR studies were used to investigate the structure of the product of the DHDPS catalysed reaction, and the data was consistent with it being 4-hydroxy-2,3,4,5- tetrahydrodipicolinic acid. Various mimics of this compound were tested as possible product inhibitors of DHDPS, and as possible substrate inhibitors of DHDPR. The most potent inhibitors of DHDPR evaluated were dipicolinic acid and isophthalic acid which both inhibited in a reversible competitive manner. Tetrahydroisophthalic acid analogues were tested for inhibition of DHDPS and DHDPR. Δ³-Tetrahydroisophthalic acid showed moderate inhibition of both enzymes, while Δ²-tetrahydroisophthalic acid showed only very slight inhibition.