Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Dual diaminopimelate biosynthesis pathways in Bacteroides fragilis and Clostridium thermocellum
Highlights
► Locus tags Bf3481 and Cthe_0922 encode authentic forms of diaminopimelate dehydrogenase. ► Bf3481 defines a previously uncharacterized diaminopimelate dehydrogenase (Ddh). ► Ddh frequently occurs in the same genome as another diaminopimelate pathway. ► Ddh has a restricted phylogenomic distribution. ► Hypothesis: Ddh evolved recently as an ancillary diaminopimelate biosynthesis mechanism.
Introduction
The diaminopimelic acid (DAP) pathway serves for the biosynthesis of lysine in bacteria, some archaea, plants and algae [1]. The pathway is also necessary for peptidoglycan biosynthesis [2]. The DAP pathway exists as four variants all of which follow the same general schema [3] (Supplementary Fig. 1). First, tetrahydrodipicolinate (THDPA) is produced from aspartate. Then THDPA is converted to meso-diaminopimelate (m-DAP). Finally, m-DAP is converted to lysine. The variant DAP pathways differ in the mechanism by which m-DAP is produced from THDPA. The most complex of the variants utilizes succinylated or acetylated intermediates and require 4 enzymes, THDPA acyltransferase (DapD) (EC:2.3.1.117), N-acyldiaminopimelate aminotransferase (DapC) (EC:2.6.1.11 and 2.6.1.17), acyl-diaminopimelate desuccinylase (DapE) (EC:3.5.1.18), and diaminopimelate epimerase (DapF) (EC:5.1.1.7) to produce m-DAP from THDPA. The other two variant pathways bypass the acylated intermediates with either a L,L-diaminopimelate aminotransferase (DapL) (EC 2.6.1.83) in conjunction with DAP epimerase (DapF) (EC 5.1.1.7) or a m-DAP dehydrogenase (Ddh) (EC 1.4.1.16).
DapL was recently described in diverse bacterial species, the Methanobacteriaceae and plants [3], [4], [5], [6], [7], [8], [9]. This enzyme catalyzes 2-oxoglutarate dependent transamination of L,L-DAP, and in most cases shows strong specificity for the L,L-DAP isomer. Genomic data shows that where it exists, DapL almost always occurs as the sole pathway for m-DAP synthesis. There are no examples in the microbial genome database of DapL being coincident in the same species with one of the acyl-DAP pathways.
Ddh catalyzes the reversible, NADP+-dependent, oxidative deamination of m-DAP. The enzyme was first discovered in Bacillus sphaericus, Corynebacterium glutamicum, and Brevibacterium sp. [10], [11], [12], [13], [14]. It is a dimer of approximately 70 kDa and shows stereo specificity for the m-DAP isomer. Ddh has weak sequence homology with other amino acid dehydrogenases. Based on structural similarity C. glutamicum Ddh resembles dihydrodipicolinate reductase (DapB) (EC:1.3.1.26), the enzyme catalyzing the preceding step in the DAP pathway, prompting Scapin et al. [15] to propose that DapB and Ddh evolved from a common ancestral enzyme.
Ddh has a restricted phylogenetic distribution. Until now it was thought to be relegated to the Firmicutes [16]. In some cases Ddh occurs as the only route for m-DAP biosynthesis. However, an example of pathway redundancy exists in C. glutamicum, which contains in addition to Ddh, the four enzymes of the acyl pathway [17]. Although the two pathways are redundant, Ddh is largely responsible for the high rate of lysine synthesis in this species [18].
In the course of their analysis of the phylogenomic distribution of DapL Hudson et al. [6] noted that Clostridium thermocellum, a Firmicute, contained a well conserved example of Ddh, suggesting that this species also has redundant pathways for DAP synthesis. Curiously, it was also noted that Bacteroides fragilis contained an ORF that might be a Ddh, but was significantly diverged from known examples of this enzyme, and moreover, this species does not belong to a phylum known to contain Ddh. Therefore, the aim of the present study was to determine whether the orthologs identified from B. fragilis and C. thermocellum are indeed functional examples of Ddh, and if so, to re-examine the phylogenomic distribution of this enzyme and to determine the frequency with which Ddh co-exists in the same species with a second pathway for DAP biosynthesis.
Section snippets
Bioinformatic methods
Orthologous protein sequences were identified by searching the microbial genome database at NCBI (http://www.ncbi.nlm.nih.gov/) with blastp using the default settings. Protein alignment was performed using ClustalW [19]. Phylogenetic trees were constructed with MEGA 4.1 using the default settings [20]. Transcriptome data from the Gene Expression Omnibus database at NCBI https://www.ncbi.nlm.nih.gov/geo/ was used to examine dap gene expression.
Orf cloning
The Ddh ortholog from B. fragilis NCTC9343 (Bf3481)
Recently identified examples of Ddh
Hudson et al. [3] found that B. fragilis and C. thermocellum contain a functional DapL, but lack the genes that define the acyl pathways. They also noted that these species appear to contain an ortholog of Ddh. In comparison to the Ddh from B. sphaericus, the first to have been described, the putative Ddh from C. thermocellum (locus tag Cthe_0922, hereafter referred to as CtDdh) shows 61.0% identity over their entire lengths, whereas the putative Ddh from B. fragilis (locus tag Bf3481,
Acknowledgment
This work was funded by USA National Science Foundation grant IPB0449542 to T.L. and C.G.; and a scholarship to P.G.M. from the FIPSE/CAPES program of the Brazilian Ministry of Education (P.G.M.). We are grateful to Dr. Sheila Patrick for contributing genomic DNA and Dr. Charles Jeffrey Smith for helpful discussions on Bacteroides fragilis gene expression data.
References (35)
- et al.
Biosynthesis of lysine in plants: evidence for a variant of the known bacterial pathways
Biochim. Biophys. Acta
(2005) - et al.
Crystal structure of LL-diaminopimelate aminotransferase from Arabidopsis thaliana: a recently discovered enzyme in the biosynthesis of l-lysine by plants and Chlamydia
J. Mol. Biol.
(2007) - et al.
Mechanism of substrate recognition and PLP-induced conformational changes in LL-diaminopimelate aminotransferase from Arabidopsis thaliana
J. Mol. Biol.
(2008) - et al.
Occurrence of meso-alpha, epsilon-diaminopimelate dehydrogenase in Bacillus sphaericus
Biochem. Biophys. Res. Commun.
(1976) N-Succinyl-alpha-amino-6-ketopimelic acid
J. Biol. Chem.
(1961)- et al.
Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set
Anal. Biochem.
(2000) - et al.
Identification and characterization of the last two unknown genes, dapC and dapF, in the succinylase branch of the l-lysine biosynthesis of Corynebacterium glutamicum
J. Biotechnol.
(2003) - et al.
Studies on the active site of succinyl-CoA:tetrahydrodipicolinate N-succinyltransferase. Characterization using analogs of tetrahydrodipicolinate
J. Biol. Chem.
(1986) - et al.
Evolutionary divergence and convergence in proteins
On biochemical evolution: lysine formation in higher plants
Proc. Natl. Acad. Sci. U. S. A.
(1959)
The cell wall peptidoglycan of Bacillus megaterium KM. I. Studies on the stereochemistry of alpha, alpha′-diaminopimelic acid
Biochemistry
An LL-diaminopimelate aminotransferase defines a novel variant of the lysine biosynthesis pathway in plants
Plant Physiol.
LL-diaminopimelate aminotransferase, a trans-kingdom enzyme of a variant diaminopimelate/lysine synthesis pathway shared by Chlamydia and plants
Proc. Natl. Acad. Sci. U. S. A.
Biochemical and phylogenetic characterization of a novel diaminopimelate biosynthesis pathway in prokaryotes identifies a diverged form of LL-diaminopimelate aminotransferase
J. Bacteriol.
Methanococci use the diaminopimelate aminotransferase (DapL) pathway for lysine biosynthesis
J. Bacteriol.
Characterization of meso-diaminopimelate dehydrogenase from Cornebacterium glutamicum and its distribution in bacteria
Agric. Biol. Chem.
The essential role of diaminopimelate dehydrogenase in the biosynthesis of lysine by Bacillus sphaericus
J. Gen. Microbiol.
Cited by (16)
The coordinated action of the enzymes in the L-lysine biosynthetic pathway and how to inhibit it for antibiotic targets
2023, Biochimica et Biophysica Acta - General SubjectsFunctional genetics of human gut commensal Bacteroides thetaiotaomicron reveals metabolic requirements for growth across environments
2021, Cell ReportsCitation Excerpt :The transaminase branch, catalyzed in two steps by DapL (BT0547) and DapF (BT0548), uses glutamate as a nitrogen source. The functions of DapL and Ddh have been verified biochemically using enzymes from B. fragilis (Hudson et al., 2008, 2011), which have 90% and 95% amino acid identity to the B. thetaiotaomicron enzymes, respectively. In Corynebacterium glutamicum, the dehydrogenase branch preferentially operates when ammonium is abundant; flux through the dehydrogenase branch is correlated with ammonium levels (Sonntag et al., 1993), and mutants in the transaminase branch require high levels of ammonium (Wehrmann et al., 1998).
Functional chararacterization of the enzymes TabB and TabD involved in tabtoxin biosynthesis by Pseudomonas syringae
2018, Biochemical and Biophysical Research CommunicationsCitation Excerpt :In our hands, we were unable to obtain pure THDPA using the reported protocol and so turned to in situ generation of THDPA from meso-DAP catalyzed by the enzyme Ddh. Physiologically, Ddh catalyzes the reductive amination of THDPA to meso-DAP; we performed the reaction in the reverse direction to convert meso-DAP to THDPA in situ [9]. In the event, when we combined THDPA with succinyl-CoA in the presence of TabB, we saw an increase in the rate of coenzyme A thiol formation as measured by Ellman assay.
- 1
Present address: Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel-Aviv University.
- 2
Present address: Departamento de Genética, Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo.