Methanogens with pseudomurein use diaminopimelate aminotransferase in lysine biosynthesis

Abstract

Methanothermobacter thermautotrophicus uses lysine for both protein synthesis and cross-linking pseudomurein in its cell wall. A diaminopimelate aminotransferase enzyme from this methanogen (MTH0052) converts tetrahydrodipicolinate to l,l-diaminopimelate, a lysine precursor. This gene complemented an Escherichia coli diaminopimelate auxotrophy, and the purified protein catalyzed the transamination of diaminopimelate to tetrahydrodipicolinate. Phylogenetic analysis indicated this gene was recruited from anaerobic Gram-positive bacteria. These results expand the family of diaminopimelate aminotransferases to a diverse set of plant, bacterial and archaeal homologs. In contrast marine methanogens from the Methanococcales, which lack pseudomurein, appear to use a different diaminopimelate pathway for lysine biosynthesis.

Figure 1

Alternative biosyntheses of meso-diaminopimelate from tetrahydrodipicolinate [8,25]. L-Tetrahydrodipicolinate exists in equilibrium with the enamine product and the acyclic L-2-oxo-6-aminopimelate product [24]. In the succinylate pathway (left), the acyclic form is protected by the tetrahydrodipicolinate N-succinyltransferase enzyme (DapD; EC 2.3.1.117). N-Succinyl-L,L-diaminopimelate aminotransferase (DapC; EC 2.6.1.17) transfers an amino group from L-glutamate, and then N-succinyl-L,L-diaminopimelate desuccinylase (DapE, EC 3.5.1.18) deprotects the product. The L,L-diaminopimelate epimerase enzyme (DapF; EC 5.1.1.7) produces meso-diaminopimelate for lysine and peptidoglycan biosynthesis. Some Gram-positive bacteria use an acetyl protecting group instead of the succinyl group. Alternatively, L,L-diaminopimelate aminotransferase (DapL; EC 2.6.1.83) directly transaminates tetrahydrodipicolinate to produce L,L-diaminopimelate. Finally, some bacteria use a diaminopimelate dehydrogenase enzyme (Ddh; EC 1.4.1.16) to reductively aminate tetrahydrodipicolinate.

Figure 2

Expression of MTH0052 complemented diaminopimelate auxotrophy in E. coli ATM769. Part A, LB agar supplemented with ampicillin (100 μg ml^-1) and arabinose (0.02% w/v) supported the growth of ATM769 (pDG428) in sector 1, ATM769 (pATM780) in sector 2, MG1655 (pBAD/HisA) in sector 3, but not ATM769 (pBAD/HisA) in sector 4. Part B, The same medium as in part A supplemented with DL-α,ε-diaminopimelate (100 μg ml^-1) supported the growth of all the strains listed for part A.

Figure 3

Reaction profile of diaminopimelate aminotransferase activity in E. coli ATM769 (pDG429) extracts expressing MTH0052 (filled circles) and E. coli ATM769 (pBAD/HisA) vector control extracts (open circles). Cell-free extract (100 μg) was incubated with 4 mM 2-oxoglutarate, 2 mM DL-α,ε-diaminopimelate (racemic), 50 μM pyridoxal 5′-phosphate, 10 mM o-aminobenzaldehyde and 100 mM Tris/HCl (pH 8) in a 300 μl reaction at 37°C. The absorbance of the yellow o-aminobenzaldehyde derivative was measured at 440 nm. Error bars show the standard deviation for three replicates of each sample.

Figure 4

Reaction products of MTH0052 derivatized with 2,4-dinitrophenylhydrazine (DNPH) and separated by reversed-phase HPLC. Curve A shows only a DNPH reagent peak from a control reaction with His₆-MTH0052 enzyme alone. Curve B shows the 2-oxoglutarate (2-OG) DNPH hydrazone from a control reaction with 2-OG and DAP substrates. Curve C shows that a reaction containing His₆-MTH0052 protein, DAP and 2-OG produced tetrahydrodipicolinate in equilibrium with 2-oxo-6-aminopimelate (OAP), which forms hydrazones with DNPH reagent.

Figure 5

Phylogram of the DapL homologs inferred by the protein maximum likelihood method. Bootstrap values from are shown near branches supported by a plurality of 100 trees. This tree is rooted using known aspartate aminotransferases enzymes and the closest homolog from M. maripaludis (MMP1527). Enzymes whose functions have been experimentally confirmed are indicated by an asterisk. The scale bar indicates one amino acid substitution per 10 positions. Sequence details are described in the Supplementary materials.