Abstract
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.
Highlights
We can test the functionality of At-diaminopimelate decarboxylase (DAPDC) enzymes by whether or not they can functionally complement the lysA mutant
We have established that the A. thaliana gene loci, At3g14390 and At5g11880, encode functional DAPDC enzymes
The recombinantly expressed eukaryotic enzymes are catalytically active but exhibit much lower activity than previously characterized bacterial orthologs. To determine whether this is a feature of plant DAPDC enzymes will require further characterization of eukaryotic DAPDC orthologs
Summary
To assess whether the identified genes annotated by the locus tags At3g14390 and At5g11880 are functional DAPDC enzymes, complementation analyses were conducted. The induced expression of At-DAPDC enzymes in lysine-free media leads to cell death because the native MurE cannot compete with the overexpressed DAPDC, and the organism is able to synthesize L-lysine, it has impaired peptidoglycan synthesis These results indicate that the two genes encode authentic DAPDC enzymes and are able to function in E. coli without the predicted chloroplast transit peptide sequences. Sedimentation velocity data at the lowest concentration (5 M) were fitted to a monomer– dimer model These analyses estimate the KD2–1 values for both At-DAPDC enzymes to be less than 1 M (Table 2). Because the rate versus enzyme concentration (Fig. 3A) is linear from 200 to 800 nM, which spans the estimated KD2–1 for both enzymes (ϳ650 nM), either both the monomer and dimer are active or the presence of substrates lowers the dissociation constant, resulting in a predominantly dimeric species at the concentration used to collect the initial rate data. The green dashed lines indicate Km values, and the red dashed lines indicate Vmax
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