Investigating the catalytic and regulatory mechanisms of dihydrodipicolinate synthase.

Dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) 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