A distinctive family of L,D-transpeptidases catalyzing L-Ala-mDAP crosslinks in Alpha- and Betaproteobacteria

Abstract

The bacterial cell-wall peptidoglycan is made of glycan strands crosslinked by short peptide stems. Crosslinks are catalyzed by DD-transpeptidases (4,3-crosslinks) and LD-transpeptidases (3,3-crosslinks). However, recent research on non-model species has revealed novel crosslink types, suggesting the existence of uncharacterized enzymes. Here, we identify an LD-transpeptidase, LDT_Go, that generates 1,3-crosslinks in the acetic-acid bacterium Gluconobacter oxydans. LDT_Go-like proteins are found in Alpha- and Betaproteobacteria lacking LD3,3-transpeptidases. In contrast with the strict specificity of typical LD- and DD-transpeptidases, LDT_Go can use non-terminal amino acid moieties for crosslinking. A high-resolution crystal structure of LDT_Go reveals unique features when compared to LD3,3-transpeptidases, including a proline-rich region that appears to limit substrate access, and a cavity accommodating both glycan chain and peptide stem from donor muropeptides. Finally, we show that DD-crosslink turnover is involved in supplying the necessary substrate for LD1,3-transpeptidation. This phenomenon underscores the interplay between distinct crosslinking mechanisms in maintaining cell wall integrity in G. oxydans.

Fig. 1. Identification of the enzyme catalyzing LD1,3-crosslink.

A Representative UV chromatogram of Gluconobacter oxydans peptidoglycan profile in stationary phase. LD1,3-crosslinked muropeptides are indicated in green. Underneath, heatmap representing the relative abundance of each muropeptide in representative bacteria species from the Acetobacteraceae family (Acetobacter pasteurianus and G. oxydans) and model organisms Escherichia coli and Vibrio cholerae. Schematic structures and nomenclature of the main muropeptides and crosslink type are shown. B Scheme of the in silico search of YkuD-domain containing enzyme candidates responsible for LD1,3-crosslinking. C Representative UV chromatograms of G. oxydans (Go) wild-type (WT), Δgox1074 and Δgox1074 pGox1074 complemented strains. LD1,3-crosslinked muropeptides are highlighted in green. D Domain analysis of Gox1074. Details of the LDT conserved motif in the YkuD domain including the catalytic Cys and His residues highlighted in yellow.

Fig. 2. Conservation of LDT_Go.

A Phylogenetic tree showing the conservation and distribution of LD-transpeptidases. In green, LDT_Go-like proteins (LD1,3-TPases) and in blue, homologs to YcbB_Ec (LD3,3-TPases). Homology was assessed by BLAST against the NCBI and OrthoDBv11 databases. B Comparison of the domains in representative LDT_Go orthologues from diverse bacterial Families: WP_011252635.1 (Gluconobacter oxydans), WP_041249327.1 (Gluconacetobacter diazotrophicus), WP_124305792.1 (Acetobacter pasteurianus), WP_099541073.1 (Acetobacter pomorum), WP_253736052.1 (Granulibacter bethesdensis), WP_254845249.1 (Desulfovibrio sp. DV), EHJ47292.1 (Solidesulfovibrio carbinoliphilus), B4EIM9_BURCJ (Burkholderia cenocepacia), QFS41266.1 (Burkholderia cepacian), WP_244096448.1 (Burkholderia dolosa), AYZ63023.1 (Burkholderia multivorans), WP_215249195.1 (Variovorax paradoxus), ALX84167.1 (Achromobacter denitrificans), AOB33672.1 (Bordetella sp. H567). The presence of a signal peptide is indicated. Signal peptides and transmembrane domains are predicted using SignalP 6.0. C UV muropeptide profiles of the heterologous expression of LDT_Go and its homologs from Acetobacter pasteurianus (LDT_Ap), Burkholderia cenocepacia (LDT_Bcn) and a catalytically inactive mutant (LDT_Bcn C354A) in E. coli BL21. LD1,3-crosslinked muropeptides are highlighted in green. D UV muropeptide profiles of G. oxydans (Go) Δldt_Go mutant and complemented derivatives expressing the LDT_Go and LDT_Bcn. LD1,3-crosslinked muropeptides are highlighted in green.

Fig. 3. LD1,3-crosslinking activity.

A In vitro activity assays of LDT_Bcn on M4-rich peptidoglycan sacculi from V. cholerae vs control (no enzyme added). LD1,3-crosslinked muropeptides are labeled in green and the products of LDT_Bcn endopeptidase activity are labeled in blue. B Muropeptide quantifications from panel (A). Variation is calculated as the difference in relative molar abundance of the muropeptide in the LDT_Bcn treated reaction minus control reaction in the in vitro assays. Relative molar abundances were calculated as the percentage of the peak area of a muropeptide, divided by its molecular weight, compared to the sum of peak areas in the chromatogram. C Effect of Ampicillin 100 µg/ml (Amp), Imipenem 100 µg/ml (Imp) and copper 1 mM (Cu²⁺) on the in vitro assays shown in panel (A). nd: not detected. D Scheme and in vitro activity assays of LDT_Bcn on M4-rich peptidoglycan sacculi previously modified with incorporated D-Met at the terminal position of the tetrapeptides. KP27 endopeptidase cleaving between L-Ala¹ and D-Glu² was used as control. Left side, UV muropeptide profiles of the mutanolysin-digested insoluble pellets showing the presence of D-Met modified muropeptides (in red), endopeptidase products (M1 and M1-M1, in blue) and LD1,3-crosslinked muropeptides (in green). The MS extracted ion chromatogram (XIC) trace of the D-Met containing tripeptide (E-mDAP-M) in the soluble fraction is shown on the right side. Error bars in graphs (B, C) represent standard deviation from mean. Source data for (B, C) are provided as a Source Data file.

Fig. 4. LDT_Go catalyzes D,L-amino acid exchange reaction.

A Scheme of the amino acid exchange reaction performed by LD3,3-TPases with non-canonical (NCDAA. e.g., D-Met) and Fluorescent (FDAA, e.g., HADA) D-amino acids. B Phase contrast (PC) and fluorescence microscopy of G. oxydans wild-type, Δldt_Go mutant and ldt_Go::ycbB_Ec allelic exchange cells labeled with HADA. Scale bar: 2 µm. Microscopy images shown are representative of three biological replicates. C Zoom-in of the UV muropeptide profiles of the same strains indicated in panel (B) cultured with 10 mM of D-Met or without (control). D Zoom-in of the UV muropeptide profile of G. oxydans wild-type highlighting the dipeptide muropeptides (M2^X, in red) including those exhibiting amino acid exchange (M2^Phe and M2^Trp), and the LD1,3-crosslinked muropeptides in green. The MS extracted ion chromatogram (XIC) traces are shown for the indicated M2^X muropeptide species. E UV muropeptide profile of G. oxydans (Go) wild-type (WT) and Δldt_Go mutant strains cultured without (control) or with 10 mM of L-Phe or D-Phe (left panel). Middle panel: zoom-in of the UV muropeptide profile where the M2^Phe muropeptide elutes. Right panel: MS extracted ion chromatogram (XIC) trace of M2^Phe.

Fig. 5. Structure of LDT_Go.

A Overview of the LDT_Go structure, colored from the N-terminus (blue) to the C-terminus (red). B Superimposition of LDT_Go (green) and the catalytic domain of YcbB_Ec (blue) bound to meropenem (yellow). Three characteristic features are highlighted, i) the capping loop of both enzymes, which in LDT_Go is disordered, ii) the LDT_Go catalytic residues C264 and H245, and iii) the distinctive LDT_Go domain within the active site composed of 2 interconnected loops between the β-strands 3 and 4 (L_β3-β4) and the β-strands 5 and 6 and (L_β5-β6). C Surface representation of LDT _Go (green) with the Pro-rich belt shown as sticks (C-atoms colored in purple). The catalytic Cys is shown in yellow. Zoom-in panels show the hydrogen bonds formed between the belt and the rest of the protein. D Superimposition of the LDT_Go structure (green) and its Alphafold2 prediction model (light brown), with the main differences highlighted in the zoom-in panels.

Fig. 6. LDT_Go active center.

Electrostatic surface representation, ranging from –13.35 to +15.88 for YcbB_Ec (A), and from –15.69 to +12.77 for LDT_Go (C) and ribbon diagram (B, D) of the donor and acceptor sites of YcbB_Ec in complex with the antibiotic meropenem (in yellow) (A, B) and of LDT_Go (C, D). The dimensions of the donor and acceptor cavities as well as the positions of the relevant loops and residues are indicated. E LDT_Go with the disaccharide tetrapeptide (M4) modeled in the active site. Molecular surface of LDT_Go (green) with the catalytic C264 highlighted in yellow. The muropeptide is represented as sticks (C atoms colored in dark red). The positions of the relevant loops are indicated by arrows and labeled. F Detailed view of the interaction of the M4 muropeptide into the LDT_Go active site. Relevant residues in the protein are represented as sticks and labeled.

Fig. 7. DD-crosslinking turnover controls LD1,3-crosslinking levels.

A Scheme of the production of monomeric substrates of LDT_Go by DD-CPases (blue) and DD-endopeptidases (red). Cleavage sites are indicated by arrowheads. DD-crosslinking (B) and LD-crosslinking (C) levels for G. oxydans (Go) wild-type (WT), Δldt_Go, Δpbp7_Go and the indicated complemented strains. Relative molar abundances of the DD- and LD1,3-crosslinked muropeptides were quantified and represented relative to the Go WT levels. D Representative images of G. oxydans WT and Δldt_Go treated with Ampicillin MIC test strips. E Table indicating MIC values for Ampicillin (Amp) and Ampicillin/Sulbactam (Amp+Sul) for the G. oxydans WT, Δldt_Go and Δldt_Go pLDT_Go complemented strain. Error bars in graphs B, C represent standard deviation from mean. Significant differences (unpaired t-test, two-tailed) are indicated: *p-value < 0.05; **p-value < 0.001; ***p-value < 0.0001; ns: not significant. Source data for B, C and exact p-values are provided as a Source Data file.