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

meso-Diaminopimelate decarboxylase catalyzes the decarboxylation of meso-diaminopimelate, the final reaction in the diaminopimelate l-lysine biosynthetic pathway. It is the only known pyridoxal-5-phosphate-dependent decarboxylase that catalyzes the removal of a carboxyl group from a d-stereocenter. Currently, only prokaryotic orthologs have been kinetically and structurally characterized. Here, using complementation and kinetic analyses of enzymes recombinantly expressed in Escherichia coli, we have functionally tested two putative eukaryotic meso-diaminopimelate decarboxylase isoforms from the plant species Arabidopsis thaliana We confirm they are both functional meso-diaminopimelate decarboxylases, although with lower activities than those previously reported for bacterial orthologs. We also report in-depth X-ray crystallographic structural analyses of each isoform at 1.9 and 2.4 Å resolution. We have captured the enzyme structure of one isoform in an asymmetric configuration, with one ligand-bound monomer and the other in an apo-form. Analytical ultracentrifugation and small-angle X-ray scattering solution studies reveal that A. thaliana meso-diaminopimelate decarboxylase adopts a homodimeric assembly. On the basis of our structural analyses, we suggest a mechanism whereby molecular interactions within the active site transduce conformational changes to the active-site loop. These conformational differences are likely to influence catalytic activity in a way that could allow for d-stereocenter selectivity of the substrate meso-diaminopimelate to facilitate the synthesis of l-lysine. In summary, the A. thaliana gene loci At3g14390 and At5g11880 encode functional. meso-diaminopimelate decarboxylase enzymes whose structures provide clues to the stereochemical control of the decarboxylation reaction catalyzed by these eukaryotic proteins.

Hard-to-place kidney offers: Donor- and system-level predictors of discard.

Understanding risk factors for deceased-donor kidney nontransplantation is important since discard rates remain high. We analyzed DonorNet® data of consecutive deceased-donor nonmandatory share primary kidney-only offers to adult candidates at our center and beyond between July 1, 2015 and March 31, 2016 for donor- and system-level risk factors of discard, defined as nontransplantation at our or subsequent transplant centers. Exclusions were hepatitis C virus/hepatitis B virus core antibody status, blood type AB, and donor <1 year based on low candidate waitlist size. Of 456 individual kidney offers, from 296 donors, 73% were discarded. Most were national (93%) offers from Kidney Donor Profile Index 35-85% (n = 233) or >85% (n = 208) donors late in the allocation sequence with prior refusals logged for numerous candidates. On multivariate regression, factors significantly associated with discard were donor cerebrovascular accident (adjusted odds ratio [aOR]: 3.32), cancer transmission concern (aOR: 6.5), renal artery luminal compromise (aOR: 3.97), biopsy score ≥3 (aOR: 5.09), 2-hour pump resistive index >0.4 (aOR: 3.27), absence of pump (aOR: 2.58), nonspecific kidney abnormality (aOR: 2.76), increasing offer cold ischemia time category 11-15, 16-20, and >21 hours (aOR: 2.07, 2.33, 2.82), nighttime notification (aOR: 2.19), and neither kidney placed at time of offer (aOR: 2.74). Many traditional determinants of discard lack discriminatory value when granular factors are assessed. System-level factors also influence discard and warrant further study.

The purification, crystallization and preliminary X-ray diffraction analysis of two isoforms of meso-diaminopimelate decarboxylase from Arabidopsis thaliana.

Diaminopimelate decarboxylase catalyses the last step in the diaminopimelate-biosynthetic pathway leading to S-lysine: the decarboxylation of meso-diaminopimelate 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.