Identification of 2, 3-dihydrodipicolinate as the product of the dihydrodipicolinate synthase reaction from Escherichia coli.

Dihydrodipicolinate synthase (DHDPS) catalyzes the first step in the pathway for the biosynthesis of L-lysine in most bacteria and plants. The substrates for the enzyme are pyruvate and L-aspartate-β-semialdehyde (ASA). The product of the reaction was originally proposed to be 2,3-dihydrodipicolinate (DHDP), but has now generally been assumed to be (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinate (HTPA). ASA is unstable at high pH and it is proposed that ASA reacts with itself. At high pH ASA also reacts with Tris buffer and both reactions are largely reversible at low pH. It is proposed that the basic un-protonated form of the amine of Tris or the α-amine of ASA reacts with the aldehyde functional group of ASA to generate an imine product. Proton NMR spectra of ASA done at different pH values shows new NMR peaks at high pH, but not at low pH, confirming the presence of reaction products for ASA at high pH. The enzymatic product of the DHDPS reaction was examined at low pH by proton NMR starting with either 3 h-pyruvate or 3 d-pyruvate and identical NMR spectra were obtained with four new NMR peaks observed at 1.5, 2.3, 3.9 and 4.1 ppm in both cases. The NMR results were most consistent with DHDP as the reaction product. The UV-spectral studies of the DHDPS reaction shows the formation of an initial product with a broad spectral peak at 254 nM. The DHDPS reaction product was further examined by reduction of the enzymatic reaction components with borohydride followed by GC-MS analysis of the mixture. Three peaks were found at 88, 119 and 169 m/z, consistent with pyruvate, homoserine (reduction product of ASA), and the reduction product of DHDP (1,2,3,6-tetrahydropyridine-2,6-dicarboxylate). There was no indication for a peak associated with the reduced form of HTPA.