The purification, crystallization and preliminary X-ray diffraction analysis of two isoforms ofmeso-diaminopimelate decarboxylase fromArabidopsis thaliana

Abstract

Diaminopimelate decarboxylase catalyses the last step in the diaminopimelatebiosynthetic pathway leading to S-lysine: the decarboxylation of mesodiaminopimelate to form S-lysine. Lysine biosynthesis occurs only in microorganisms and plants, and lysine is essential for the growth and development of animals. Thus, the diaminopimelate pathway represents an attractive target for antimicrobial and herbicide treatments and has received considerable attention from both a mechanistic and a structural viewpoint. Diaminopimelate decarboxylase has only been characterized in prokaryotic species. This communication describes the first structural studies of two diaminopimelate decarboxylase isoforms from a plant. The Arabidopsis thaliana diaminopimelate decarboxylase cDNAs At3g14390 (encoding DapDc1) and At5g11880 (encoding DapDc2) were cloned from genomic DNA and the recombinant proteins were expressed and purified from Escherichia coli Rosetta (DE3) cells. The crystals of DapDc1 and DapDc2 diffracted to beyond 2.00 and 2.27 Å resolution, respectively. Understanding the structural biology of diaminopimelate decarboxylase from a eukaryotic species will provide insights for the development of future herbicide treatments, in particular.

Structure–function analyses of two plant meso-diaminopimelate decarboxylase isoforms reveal that active-site gating provides stereochemical control

“…It should be noted that previous work by our group shows that both At-DAPDC isoforms were able to complement another lysA mutant in a study that surveyed the lysine anabolic genes from A. thaliana (12). However, the previous analysis used fulllength cDNAs for both proteins, although the analysis reported here was completed without the sequences that encode the predicted transit peptide that denotes localization to the chloroplast (13).…”

“…At-DAPDC1 and At-DAPDC2 were crystallized as reported previously (13). All relevant data-collection and processing parameters are given in Table 3.…”

“…The cDNAs At3g14390 (At-DAPDC1) and At5g11880 (At-DAPDC2), encoding the two isoforms of DAPDC from A. thaliana, were cloned as described previously (13). The forward and reverse primers used to amplify the At3g14390 cDNA were 5Ј-CCCCCGGATCCATGGCCGCCGTCGTTTCTCAAAA-CTCGTCC-3Ј and 5Ј-CCCCCGTCGACTCATAGACCTTC-…”

“…Purified At-DAPDC1 and At-DAPDC2 were crystallized and X-ray diffraction data collected as reported previously (13). The crystal structures were refined using REFMAC (28) with iterative rounds of model building performed in COOT (29).…”

“…Thus far, the only studies that have been reported for a eukary-otic DAPDC are of the two Arabidopsis thaliana orthologs that were initially identified and verified through a functional complementation assay (12). Preliminary structural studies have been carried out on these orthologs, but the quaternary association has not yet been investigated (13).…”