Investigating the catalytic and regulatory mechanisms of dihydrodipicolinate synthase.
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Dihydrodipicolinate synthase (DHDPS, E.C. 126.96.36.199) catalyses the branchpoint reaction of lysine biosynthesis in plants and microbes-the condensation of (S)-aspartate-β-semialdehyde ((S)-ASA) and pyruvate. In an attempt to better understand the reaction catalysed by DHDPS, the wild-type enzyme was overexpressed and, following purification, kinetically characterised using a coupled assay. The kinetic mechanism was of the ping-pong type and the lunetic parameters obtained were consistent with other literature values. An improved synthesis of (S)-ASA was successfully achieved in three steps with an overall yield of 94%; this represents a significant advance over previously published routes to (S)-ASA. There are literature reports that high levels of (S)-ASA inhibit DHDPS, whilst others have not observed this phenomenon. It is shown unequivocally that this difference can be attributed to the different methods of preparing (S)-ASA used by each researcher: DHDPS is not inhibited by (S)-ASA, rather, the inhibition is due to an, as yet, unidentified inhibitor in the preparations of the substrate generated by ozonolysis. Others have published the crystal structure of wild-type DHDPS to 2.5-Å. They have hypothesized that the catalytic mechanism of the enzyme involves a catalytic triad of amino acid residues, Y133, T44, and Y107 that provides a proton-relay to transport protons within the active site and from the active site to solvent. Additionally, R138 has been implicated in (S)-ASA binding. These hypotheses were tested using site-directed mutagenesis to produce five mutant enzymes: DHDPSY133F, DHDPS-T44S, DHDPS-R138H, DHDPS-T44V, and DHDPS-Y107F. Each of these mutants had reduced catalytic activity, consistent with the catalytic triad hypothesis. DHDPSR138H showed an increased KmASA, consistent with its role in (S)-ASA binding. The crystal structures of DHDPS-Y133F, DHDPS-T44V, DHDPS-Y107F were determined to at least 2.35-Å resolution and compared to the wild-type structure. All mutant enzymes crystallised into the same space group as the wild-type and only minor differences in structure were observed. These results suggest that the catalytic triad is indeed in operation in wild-type DHDPS. The mechanism of lysine inhibition in DHDPS appears complex but two hypotheses were previously suggested. These were that lysine affects the proton-relay and/or the flexibility of R138 to inhibit DHDPS catalysis. The mutants generated above were used to test these hypothesises. DHDPSY133F, DHDPS-T44V, and DHDPS-R138H showed less sensitivity to lysine inhibition compared to the wild-type, while DHDPS-T44S and DHDPS-Y107F showed identical behaviour to the wild-type. The results showed that some mutations in the proton-relay attenuated lysine inhibition so lysine may operate, at least in part, via this motif. That DHDPS-R138H also showed decreased sensitivity to lysine suggests that this residue also has some role in lysine inhibition. However, the crystal structure of DHDPS-T44V with bound lysine showed that the flexibility of R138 had increased, in contrast to the situation of the wild-type. To reconcile these results, a new mechanism of inhibition is proposed involving a hitherto undocumented channel of well-defined water molecules.