Methanococci use the diaminopimelate aminotransferase (DapL) pathway for lysine biosynthesis.

Two lysine biosynthesis pathways evolved separately in organisms, the diaminopimelic acid (DAP) and aminoadipic acid (AAA) pathways. The DAP pathway synthesizes l-lysine from aspartate and pyruvate, and diaminopimelic acid is an intermediate. This pathway is utilized by most bacteria, some archaea, some fungi, some algae, and plants (28, 29). The AAA pathway synthesizes l-lysine from α-ketoglutarate and acetyl coenzyme A (acetyl-CoA), and α-aminoadipic acid is an intermediate. This pathway is utilized by most fungi, some algae, the bacterium Thermus thermophilus, and probably some archaea, such as Sulfolobus, Thermoproteus, and Pyrococcus (27, 36). No organism is known to possess both pathways. There are four known variations of the DAP pathway in bacteria: the succinylase, acetylase, aminotransferase, and dehydrogenase pathways (Fig. ​(Fig.1).1). These pathways share the steps converting l-aspartate to l-2,3,4,5-tetrahydrodipicolinate (THDPA), but the subsequent steps leading to the production of meso-diaminopimelate, the immediate precursor of l-lysine, are different. The succinylase pathway acylates THDPA with succinyl-CoA to generate N-succinyl-ll-2-amino-6-ketopimelate and forms meso-DAP by subsequent transamination, desuccinylation, and epimerization. This pathway is utilized by proteobacteria and many firmicutes and actinobacteria (12, 14, 20, 29). The acetylase pathway is analogous to the succinylase pathway but uses N-acetyl intermediates. This pathway is limited to certain Bacillus species, in which the corresponding genes have not been identified (33, 39). The aminotransferase pathway converts THDPA directly to ll-DAP by diaminopimelate aminotransferase (DapL) without acylation. This pathway is shared by cyanobacteria (19), chlamydia (24), the archaeon Methanothermobacter thermautotrophicus (15, 18), and the plant Arabidopsis thaliana (19). The dehydrogenase pathway forms meso-DAP directly from THDPA, NADPH, and NH4+ by using diaminopimelate dehydrogenase (Ddh). This pathway is utilized by some Bacillus and Brevibacterium species and Corynebacterium glutamicum (25, 26, 40). Most bacteria use only one of the four variants, although certain bacteria, such as C. glutamicum and Bacillus macerans, possess both the succinylase and dehydrogenase pathways (3, 30). FIG. 1. Variations in the DAP pathway for lysine biosynthesis. 1, succinylase pathway; 2, acetylase pathway; 3, aminotransferase pathway; 4, dehydrogenase pathway. Abbreviations and designations: THDPA, l-2,3,4,5-tetrahydrodipicolinate; l,l-DAP, ll-2,6-diaminopimelate; ... The diaminopimelate aminotransferase (DapL) catalyzes the transfer of an amino group from l-glutamate to THDPA, forming ll-DAP (19, 24). It uses pyridoxal 5′-phosphate (PLP) as a coenzyme and has constrained substrate specificity. DapL is not closely related to the DapC/ArgD aminotransferase, which functions in the succinylase pathway. Comparative genomic analysis identified dapL homologs in both bacterial and archaeal genomes. Homologs of dapD and dapE have not been found in genomes with dapL homologs, suggesting that transamination of THDPA does not require succinylation in these organisms (18). Phylogenetic analysis also suggested classification of DapL into two groups, DapL1 and DapL2, which share ∼30% amino acid sequence identity (18). These two groups both exhibit DapL activity, and they cannot be differentiated by kinetic properties (18, 37). The distribution of the two groups is not obviously associated with specific prokaryotic lineages (18). Methanogens are strictly anaerobic archaea that obtain all or most of their energy for growth from the production of large quantities of methane. All methanogens belong to the Euryarchaeota and are currently classified in six orders: Methanobacteriales, Methanococcales, Methanomicrobiales, Methanosarcinales, Methanopyrales, and Methanocellales (23, 41, 42). Biochemical studies of Methanocaldococcus jannaschii and Methanococcus voltae belonging to Methanococcales, Methanospirillum hungatei belonging to Methanomicrobiales, and Methanothermobacter thermautotrophicus belonging to Methanobacteriales suggested that these organisms derive their l-lysine from a DAP pathway, but the studies did not discriminate among the four DAP pathway variations (2, 9, 10, 32). Genome sequence analysis also suggested a DAP pathway in Methanosarcina mazei belonging to Methanosarcinales (8). Recent studies identified a dapL homolog belonging to the DapL1 group in M. thermautotrophicus. The gene product complemented an Escherichia coli dapD dapE double mutant and catalyzed the transamination of DAP to THDPA, suggesting that Methanobacteriales use the DapL pathway for l-lysine biosynthesis (15, 18). Homologs of asd, dapA, dapB, dapF, and lysA have been identified in the genomes of M. maripaludis and M. jannaschii belonging to the Methanococcales, but homologs responsible for the conversion of THDPA to ll-DAP have not been annotated (4, 17). Here we identified methanococcal DapL homologs and demonstrated that the DapL pathway is present in Methanococcales.