Structure of the 4-hydroxy-tetrahydrodipicolinate synthase from the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV and the phylogeny of the aminotransferase pathway

Abstract

The enzyme 4-hydroxy-tetrahydrodipicolinate synthase (DapA) is involved in the production of lysine and precursor molecules for peptidoglycan synthesis. In a multistep reaction, DapA converts pyruvate and L-aspartate-4-semialdehyde to 4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid. In many organisms, lysine binds allosterically to DapA, causing negative feedback, thus making the enzyme an important regulatory component of the pathway. Here, the 2.1 Å resolution crystal structure of DapA from the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV is reported. The enzyme crystallized as a contaminant of a protein preparation from native biomass. Genome analysis reveals that M. fumariolicum SolV utilizes the recently discovered aminotransferase pathway for lysine biosynthesis. Phylogenetic analyses of the genes involved in this pathway shed new light on the distribution of this pathway across the three domains of life.

Keywords: 4-hydroxy-tetrahydrodipicolinate synthase; Methylacidiphilum fumariolicum SolV; aminotransferase pathway; methanotroph.

Figure 1

The multistep reaction catalysed by DapA. The catalytic Lys162 is condensed with pyruvate, forming an enamine that reacts with l-aspartic semialdehyde (ASA). The resulting covalent intermediate is cyclized, resulting in the production of (2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid (HTPA).

Figure 2

A Tris–glycine 4–20% gradient gel (Coomassie staining) of the protein preparation (lane H). Bands H1–H7 were excised for peptide mass fingerprinting. Band H4 was identified as M. fumariolicum SolV DapA (MfumV2_0415), bands H1 and H3 as the large and small subunits of the [NiFe] hydrogenase, respectively (MfumV2_0979 and MfumV2_0978), and band H2 as a degradation product of the large subunit. Band H5 was identified as elongation factor Ts (MfumV2_2094) and band H7 as the ClpB chaperone (MfumV2_0146). No match was found for band H6. Lane MW contains a molecular-weight ladder, with the molecular masses indicated in kDa on the left.

Figure 3

Overview of the DapA tetramer from M. fumariolicum SolV. The four monomers are coloured various shades of green. The right panel shows a cut through the tetramer. In two of the four monomers, the positions of the active sites (red), as well as the locations corresponding to the lysine-binding sites for allosteric regulation (yellow), are indicated.

Figure 4

(a) Stereo figure showing the active site of M. fumariolicum SolV (shades of green) superimposed with that of E. coli DapA (light blue/grey). A sulfate (or phosphate; see text) ion is bound in the active site. The residues of the catalytic triad, as well as the catalytic lysine, superimpose closely. (b) Stereo figure showing the putative allosteric pocket in M. fumariolicum SolV (shades of green) superimposed with that in the lysine-inhibited C. jejuni DapA structure (dark red/brown). The residues interacting with the allosteric lysine (Lys) in the C. jejuni structure are conserved in M. fumariolicum SolV DapA, including His56, which is believed to be diagnostic for allosteric regulation. Residues from an adjacent subunit are marked with an apostrophe.

Figure 5

Superposition of DapA from M. fumariolicum SolV (SolV, green) with DapA from C. jejuni without (C. jejuni, blue) and with (C. jejuni + Lys, red) lysine in the allosteric pocket. The protein is shown as a cartoon; the covalent adduct in the active site (Act.) and the lysine in the allosteric pocket (All.) are shown as sticks. The approximate boundary between the domains is indicated by the dashed line. Domain 1 of the C. jejuni structure was used for superposition. Domain 2 of the SolV protein superimposes closely with the corresponding domain in the C. jejuni protein without lysine in the allosteric site and less well with the structure of the C. jejuni enzyme with a lysine bound in the allosteric pocket.

Figure 6

Stereofigure showing the cysteine residues at the allosteric interface in DapA from M. fumariolicum SolV (green) and A. aeolicus (brown/yellow). In the A. aeolicus enzyme the residues form an intersubunit disulfide bond, whereas in M. fumariolicum DapA the side chains point away from each other.

Figure 7

(a) Distribution of DapL among Eukarya, Archaea and Bacteria according to sequences present in the UniProt database. The total number of sequences included in each bar is shown in parentheses. The grey bar represents all bacterial phyla that individually account for less than 2% of the total number of bacterial DapL sequences (14 phyla in total). DPANN: archaeal superphylum consisting of Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota and Nanohaloarchaeota. TACK: archaeal superphylum consisting of Thaumarchaeota, Aigarchaeota, Crenarchaeota and Korarchaeota. (b) Neighbour-joining phylogenetic trees of DapA (left) and DapL (right), showing the relationships between methanotrophic and non-methanotrophic Verrucomicrobia, DapL-containing methanotrophic Proteobacteria, methanogenic Euryarchaeota, Asgardarchaeota and Eukarya. Bracketed numbers indicate the number of sequences in a collapsed branch. Both trees contain sequences from the same taxa. For accession numbers, see Supplementary Table S1.