Genomic and Biochemical Analysis of the Diaminopimelate and Lysine Biosynthesis Pathway in Verrucomicrobium spinosum: Identification and Partial Characterization of L,L-Diaminopimelate Aminotransferase and UDP-N-Acetylmuramoylalanyl-D-glutamyl-2,6-meso-Diaminopimelate Ligase

Abstract

The Gram-negative bacterium Verrucomicrobium spinosum has attracted interest in recent years following the sequencing and annotation of its genome. Comparative genomic analysis of V. spinosum using diaminopimelate/lysine metabolic genes from Chlamydia trachomatis suggests that V. spinosum employs the L,L-diaminopimelate aminotransferase (DapL) pathway for diaminopimelate/lysine biosynthesis. The open reading frame corresponding to the putative dapL ortholog was cloned and the recombinant enzyme was shown to possess L,L-diaminopimelate aminotransferase activity in vitro. In vivo analysis using functional complementation confirmed that the dapL ortholog was able to functionally complement an E. coli mutant that confers auxotrophy for diaminopimelate and lysine. In addition to its role in lysine biosynthesis, the intermediate diaminopimelate has an integral role in peptidoglycan biosynthesis. To this end, the UDP-N-acetylmuramoylalanyl-d-glutamyl-2,6-meso-diaminopimelate ligase ortholog was also identified, cloned, and was shown to possess meso-diaminopimelate ligase activity in vivo. The L,L-diaminopimelate aminotransferase pathway has been experimentally confirmed in several bacteria, some of which are deemed pathogenic to animals. Since animals, and particularly humans, lack the genetic machinery for the synthesis of diaminopimelate/lysine de novo, the enzymes involved in this pathway are attractive targets for development of antibiotics. Whether dapL is an essential gene in any bacteria is currently not known. V. spinosum is an excellent candidate to investigate the essentiality of dapL, since the bacterium employs the DapL pathway for lysine and cell wall biosynthesis, is non-pathogenic to humans, facile to grow, and can be genetically manipulated.

Biochemical Characterization of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: meso-2,6-diaminopimelate ligase (MurE) from Verrucomicrobium spinosum DSM 4136T

“…A recent study from our laboratories confirmed the presence of the plant-like biosynthetic pathway for diaminopimelate and l -lysine in V. spinosum through the partial characterization of the enzyme l , l -diaminopimelate aminotransferase (DapL) [10]. In the same study, we identified the MurE ortholog and showed that the enzyme was able to functionally complement an E. coli mutant that harbors a mutation in the murE gene [10].…”

“…In the same study, we identified the MurE ortholog and showed that the enzyme was able to functionally complement an E. coli mutant that harbors a mutation in the murE gene [10].…”

“…The BlastP algorithm from the Integrated Microbial Genomes (IMG) database was employed. The search resulted in the identification of a putative MurE from V. spinosum annotated by the locus tag VspiD_010100019130, which is 37% identical to the C. trachomatis MurE [10].…”

“…Importantly, the bacterium is not pathogenic to mammals based on what we currently know. Since the genome of the organism can be genetically modified using transposon mutagenesis, analysis of genes that are essential for V. spinosum that are involved in the diaminopimelate/ l -lysine biosynthesis can be the focus of future studies [10], [35].…”

“…V. spinosum was found to employ the recently discovered l , l -diaminopimelate aminotransferase (DapL) pathway [6], [7], [8], [9] as the sole route for the synthesis of diaminopimelate (A 2 pm) and l -lysine ( l -Lys), based on biochemical and bioinformatical evidence [10]. In the anabolism of PG, the penultimate intermediate in the l -lysine biosynthesis pathway, meso -diaminopimelate ( meso -A 2 pm), serves as one of the cross-linking amino acids in Gram-negative bacteria, and l -Lys serves the same purpose in many Gram-positive bacteria [11].…”