Dual diaminopimelate biosynthesis pathways in Bacteroides fragilis and Clostridium thermocellum

https://doi.org/10.1016/j.bbapap.2011.04.019Get rights and content

Abstract

Bacteroides fragilis and Clostridium thermocellum were recently found to synthesize diaminopimelate (DAP) by way of LL-DAP aminotransferase. Both species also contain an ortholog of meso-diaminopimelate dehydrogenase (Ddh), suggesting that they may have redundant pathways for DAP biosynthesis. The B. fragilis Ddh ortholog shows low homology with other examples of Ddh and this species belongs to a phylum, the Bacteriodetes, not previously known to contain this enzyme. By contrast, the C. thermocellum ortholog is well conserved with known examples of Ddh. Using in vitro and in vivo assays both the B. fragilis and C. thermocellum enzymes were found to be authentic examples of Ddh, displaying kinetic properties typical of this enzyme. The result indicates that B. fragilis contains a sequence diverged form of Ddh. Phylogenomic analysis of the microbial genome database revealed that 77% of species with a Ddh ortholog also contain a second pathway for DAP biosynthesis suggesting that Ddh evolved as an ancillary mechanism for DAP biosynthesis.

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.

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    Present address: Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel-Aviv University.

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