Characterization of a Bordetella pertussis Diaminopimelate (DAP) Biosynthesis Locus Identifies dapC , a Novel Gene Coding for an N -Succinyl- l,l -DAP Aminotransferase

Abstract

The functional complementation of two Escherichia coli strains defective in the succinylase pathway of meso-diaminopimelate (meso-DAP) biosynthesis with a Bordetella pertussis gene library resulted in the isolation of a putative dap operon containing three open reading frames (ORFs). In line with the successful complementation of the E. coli dapD and dapE mutants, the deduced amino acid sequences of two ORFs revealed significant sequence similarities with the DapD and DapE proteins of E. coli and many other bacteria which exhibit tetrahydrodipicolinate succinylase and N-succinyl-L,L-DAP desuccinylase activity, respectively. The first ORF within the operon showed significant sequence similarities with transaminases and contains the char- (29): (i) the dehydrogenase variant in which the intermediate tetrahydrodipicolinate (THDP) common to all three pathways is converted in a single step to DAP, (ii) the succinylase variant involving two succinylated intermediates, and (iii) the acetylase variant using the acetyl residue instead of succinyl as the blocking group (Fig. 1).In the succinylase pathway, THDP is converted by the succinyltransferase (DapD) to N-succinyl-2-amino-6-ketopimelate, which is the substrate of the aminotransferase DapC. Its product, N-succinyl-L,L-DAP, is converted by DapE, a desuccinylase, to the common product of both the succinylase and acetylase pathways, L,L-DAP (27). The acetylase and/or dehydrogenase pathways are found among members of the genus Bacillus (38), while the succinylase pathway is present in Escherichia coli (18). In Corynebacterium glutamicum, both the succinylase and dehydrogenase pathways can operate in D,L-DAP and L-lysine biosynthesis (30). This high variability and flexibility of DAP pathways might ensure the availability of a sufficient amount of meso-DAP for cell wall synthesis under different environmental conditions (37). In addition to the vital role of DAP in the cross-linking of the glycan backbones in the bacterial cell wall and in providing lysine for protein biosynthesis, DAP is a central constituent in the Bordetella pertussis tracheal cytotoxin, which is an important virulence factor that causes several pathological effects in epithelial cells (7,21).Since DAP is neither produced nor required by humans, many efforts have been made to study DAP biosynthetic enzymes (8,24,28), and DAP analogs are evaluated for their potential to inhibit bacterial growth. Furthermore, the use of DAP auxotrophic mutants of Mycobacterium tuberculosis, Mycobacterium bovis BCG, Salmonella subspecies, or Helicobacter pylori as attenuated vaccine strains or for the maintenance of cloning vectors expressing foreign antigens in such attenuated strains has been proposed (9, 17, 23). Although the biochemistry of the DAP-lysine pathway is very well understood, the genes encoding enzymes involved in this pathway have not been completely characterized. Indeed, only three out of the four genes required for the succinyl pathway of E. coli, dapD, dapE, and dapF, encoding THDP succinylase, and ...

Methanococci Use the Diaminopimelate Aminotransferase (DapL) Pathway for Lysine Biosynthesis

“…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.…”

Enzymology of Bacterial Lysine Biosynthesis

“…The gene encoding NSDAP-AT (dapC), is found in a large number of bacterial species including Bordetella pertussis (Fuchs et al, 2000), C. glutamicum (Hartmann et al, 2003), E. coli, (Peterkofsky & Gilvarg., 1961) and M. tuberculosis (Weyand et al, 2006). In E. coli, the gene encoding NSDAP-AT is annotated argD (Ledwidge & Blanchard., 1999).…”

Biochemical and Phylogenetic Characterization of a Novel Diaminopimelate Biosynthesis Pathway in Prokaryotes Identifies a Diverged Form of ll -Diaminopimelate Aminotransferase

“…The tree was constructed by comparing representatives from DapL1 (those more closely related to sll0480) and DapL2 (more closely related to Sfum_0054) with a selection of other aminotransferases. Several examples of DapC and ArgD were included because these enzymes catalyze transamination in the acyl-DAP pathways (8,12,17), the reaction that is analogous to that catalyzed by DapL. Several examples of AspC and TyrB were included because these represent the prototype of class I/II aminotransferases.…”

“…Based upon its constrained substrate specificity, DapL does not appear to be closely related to the DapC aminotransferase that functions in the acyl-DAP pathways. In fact, at least three different and divergent aminotransferases are known to catalyze the DapC reaction (6,8,12,17,18). The crystal structure of the DapL protein from Arabidopsis thaliana has been solved, providing insight into the substrate specificity of the enzyme (35).…”