Outline of pathways of synthesis of essential amino acids.
tabaci BR2.024 for lysine and tabtoxinine-beta-lactam biosynthesis.
Overproduction of (S)-lysine in plants is currently carried out through the expression of a feedback insensitive DHDPS enzyme, such as that from Corynebacterium glutamicum. An increased understanding of how plant DHDPS is inhibited by (S)-lysine opens the potential for the generation of a lysine insensitive plant variant for use in improved crop quality. At-DHDPS2 is tightly regulated through inhibition by very low concentrations of (S)-lysine, the end product of the pathway. However, unlike a previous study of the Ns-DHDPS enzyme that observed changes in the orientation of the subunits upon binding of (S)-lysine, X-ray crystallographic and small angle X-ray scattering studies showed that no large structural changes occurred upon binding of (S)-lysine to the allosteric site of At-DHDPS2. Indeed, the changes observed in the lysine bound crystal structure compared to the unliganded structure are confined to residues of the lysine binding pocket itself. The side chain of Trp116 undergoes a shift in rotamer upon lysine binding, closing down against the side chain of the bound lysine in a gate like action. Small rearrangements of the Glu147, His119, Ile120 and Arg146 side chains to accommodate the lysine molecule were also observed. However, these changes in side chain configurations were not propagated to other regions of the structure. We note that the resolution of the structures presented here (2.0–2.2 A) is considerably higher than that of the Ns-DHDPS structures previously published (∼2.8 A) . Since the lysine binding sites of At-DHDPS2 are not situated close to crystal contacts, crystal packing arguments should not preclude observation of structural changes around this site. As such, we would expect to detect any significant conformational changes in the structure due to lysine binding.
The enzymology of lysine biosynthesis in higher plants.
From the practical point of view, this work, in addition to our previous analyses of the vitamin-specific regulons (–), demonstrate one more example of the power of comparative genomics for the functional gene annotation. Comparative analysis of pathway-specific regulatory sites in bacterial genomes is very effective in this respect. Combination of genomic techniques allowed us to identify candidates for previously missing lysine biosynthetic and transport genes in a variety of bacterial species.