Although lysine (Lys) biosynthesis in plants is known to occur by way of a pathway that utilizes diaminopimelic acid (DAP) as a central intermediate, the available evidence suggests that none of the known DAP-pathway variants found in nature occur in plants. A new Lys biosynthesis pathway has been identified in Arabidopsis (Arabidopsis thaliana) that utilizes a novel transaminase that specifically catalyzes the interconversion of tetrahydrodipicolinate and LL-diaminopimelate, a reaction requiring three enzymes in the DAP-pathway variant found in Escherichia coli. The LL-DAP aminotransferase encoded by locus At4g33680 was able to complement the dapD and dapE mutants of E. coli. This result, in conjunction with the kinetic properties and substrate specificity of the enzyme, indicated that LL-DAP aminotransferase functions in the Lys biosynthetic direction under in vivo conditions. Orthologs of At4g33680 were identified in all the cyanobacterial species whose genomes have been sequenced. The Synechocystis sp. ortholog encoded by locus sll0480 showed the same functional properties as At4g33680. These results demonstrate that the Lys biosynthesis pathway in plants and cyanobacteria is distinct from the pathways that have so far been defined in microorganisms.

The synthesis of meso-diaminopimelic acid (m-DAP) in bacteria is essential for both peptidoglycan and lysine biosynthesis. From genome sequencing data, it was unclear how bacteria of the Chlamydiales order would synthesize m-DAP in the absence of dapD, dapC, and dapE, which are missing from the genome. Here, we assessed the biochemical capacity of Chlamydia trachomatis serovar L2 to synthesize m-DAP. Expression of the chlamydial asd, dapB, and dapF genes in the respective Escherichia coli m-DAP auxotrophic mutants restored the mutants to DAP prototrophy. Screening of a C. trachomatis genomic library in an E. coli ⌬dapD DAP auxotroph identified ct390 as encoding an enzyme that restored growth to the Escherichia coli mutant. ct390 also was able to complement an E. coli ⌬dapD ⌬dapE, but not a ⌬dapD ⌬dapF mutant, providing genetic evidence that it encodes an aminotransferase that may directly convert tetrahydrodipicolinate to L,Ldiaminopimelic acid. This hypothesis was supported by in vitro kinetic analysis of the CT390 protein and the fact that similar properties were demonstrated for the Protochlamydia amoebophila homologue, PC0685. In vivo, the C. trachomatis m-DAP synthesis genes are expressed as early as 8 h after infection. An aminotransferase activity analogous to CT390 recently has been characterized in plants and cyanobacteria. This previously undescribed pathway for m-DAP synthesis supports an evolutionary relationship among the chlamydiae, cyanobacteria, and plants and strengthens the argument that chlamydiae synthesize a cell wall despite the inability of efforts to date to detect peptidoglycan in these organisms.Chlamydophila ͉ meso-diaminopimelic acid biosynthesis ͉ peptidoglycan ͉ Nod1 ͉ pathway holes T he synthesis of meso-diaminopimelic acid (m-DAP) is crucial for survival of most bacteria. m-DAP is the direct precursor of lysine, an amino acid essential for protein synthesis. Furthermore, m-DAP and lysine play pivotal roles in peptidoglycan (PG) synthesis by cross-linking PG glycan chains to provide strength and rigidity to the PG (1). Plants also synthesize lysine via the m-DAP pathway (2, 3). In contrast, mammalian cells neither synthesize nor use m-DAP as a substrate in any metabolic pathway, and lysine is an essential amino acid that is obtained from dietary sources (4 -6). The absence of an m-DAP͞lysine synthesis pathway in mammalian cells makes the enzymes of the bacterial pathway attractive targets for antimicrobial therapy.m-DAP͞lysine synthesis comprises a branch of the aspartate metabolic pathway that also includes the synthesis of methionine, threonine, and isoleucine (Fig. 1). Common to the synthesis of all these amino acids is the conversion of L-aspartate to L-aspartate-semialdehyde via LysC and Asd (7,8). The first reaction unique to m-DAP͞lysine synthesis is the DapAcatalyzed condensation of L-aspartate-semialdehyde and pyruvate to generate dihydrodipicolinate, which is reduced subsequently by DapB to tetrahydrodipicolinate (THDP). Hereafter, we refer to the four-step synthesis of T...

A variant of the diaminopimelate (DAP)-lysine biosynthesis pathway uses an LL-DAP aminotransferase (DapL, EC 2.6.1.83) to catalyze the direct conversion of L-2,3,4,5-tetrahydrodipicolinate to LL-DAP. Comparative genomic analysis and experimental verification of DapL candidates revealed the existence of two diverged forms of DapL (DapL1 and DapL2). DapL orthologs were identified in eubacteria and archaea. In some species the corresponding dapL gene was found to lie in genomic contiguity with other dap genes, suggestive of a polycistronic structure. The DapL candidate enzymes were found to cluster into two classes sharing approximately 30% amino acid identity. The function of selected enzymes from each class was studied. Both classes were able to functionally complement Escherichia coli dapD and dapE mutants and to catalyze LL-DAP transamination, providing functional evidence for a role in DAP/lysine biosynthesis. In all cases the occurrence of dapL in a species correlated with the absence of genes for dapD and dapE representing the acyl DAP pathway variants, and only in a few cases was dapL coincident with ddh encoding meso-DAP dehydrogenase. The results indicate that the DapL pathway is restricted to specific lineages of eubacteria including the Cyanobacteria, Desulfuromonadales, Firmicutes, Bacteroidetes, Chlamydiae, Spirochaeta, and Chloroflexi and two archaeal groups, the Methanobacteriaceae and Archaeoglobaceae.meso-Diaminopimelate (m-DAP) is the immediate precursor of lysine in prokaryotes and plants (3,25), and in many eubacteria, m-DAP is also required for the synthesis of murein (34). In addition to the m-DAP pathway, another, completely different method has evolved for the biosynthesis of lysine (33) involving the intermediate compound ␣-amino adipic acid. The ␣-amino adipic acid pathway is found in most fungi (32), and a modification of it is found in selected eubacterial and archaeal species (21).Four different variants of the m-DAP-lysine pathway have been discerned, and they are depicted in Fig. 1. All share the initial and terminal steps but differ in the reactions at the center of the pathway. The common reactions include the first, in which aspartate ␤-semialdehyde is condensed with pyruvate to produce dihydrodipicolinate; the second, in which dihydrodipicolinate is reduced to L-2,3,4,5-tetrahydrodipicolinate (THDPA); and the final reaction, in which m-DAP is decarboxylated to form lysine. The enzymes catalyzing these reactions, dihydrodipicolinate synthase (DapA, EC 4.2