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. 2023 Oct 17;62(20):2970-2981.
doi: 10.1021/acs.biochem.3c00293. Epub 2023 Oct 2.

Structure and Function of ArnD. A Deformylase Essential for Lipid A Modification with 4-Amino-4-deoxy-l-arabinose and Polymyxin Resistance

Daniel Muñoz-Escudero  1 , Steven D Breazeale  2 , Myeongseon Lee  3 , Ziqiang Guan  2 , Christian R H Raetz  2 , Marcelo C Sousa  3
Affiliations

Structure and Function of ArnD. A Deformylase Essential for Lipid A Modification with 4-Amino-4-deoxy-l-arabinose and Polymyxin Resistance

Daniel Muñoz-Escudero et al. Biochemistry. .

Abstract

Covalent modification of lipid A with 4-deoxy-4-amino-l-arabinose (Ara4N) mediates resistance to cationic antimicrobial peptides and polymyxin antibiotics in Gram-negative bacteria. The proteins required for Ara4N biosynthesis are encoded in the pmrE and arnBCADTEF loci, with ArnT ultimately transferring the amino sugar from undecaprenyl-phospho-4-deoxy-4-amino-l-arabinose (C55P-Ara4N) to lipid A. However, Ara4N is N-formylated prior to its transfer to undecaprenyl-phosphate by ArnC, requiring a deformylase activity downstream in the pathway to generate the final C55P-Ara4N donor. Here, we show that deletion of the arnD gene in an Escherichia coli mutant that constitutively expresses the arnBCADTEF operon leads to accumulation of the formylated ArnC product undecaprenyl-phospho-4-deoxy-4-formamido-l-arabinose (C55P-Ara4FN), suggesting that ArnD is the downstream deformylase. Purification of Salmonella typhimurium ArnD (stArnD) shows that it is membrane-associated. We present the crystal structure of stArnD revealing a NodB homology domain structure characteristic of the metal-dependent carbohydrate esterase family 4 (CE4). However, ArnD displays several distinct features: a 44 amino acid insertion, a C-terminal extension in the NodB fold, and sequence divergence in the five motifs that define the CE4 family, suggesting that ArnD represents a new family of carbohydrate esterases. The insertion is responsible for membrane association as its deletion results in a soluble ArnD variant. The active site retains a metal coordination H-H-D triad, and in the presence of Co2+ or Mn2+, purified stArnD efficiently deformylates C55P-Ara4FN confirming its role in Ara4N biosynthesis. Mutations D9N and H233Y completely inactivate stArnD implicating these two residues in a metal-assisted acid-base catalytic mechanism.

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Conflict of interest statement

Conflict of interest statement.

The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1:
Figure 1:. Negative ion ESI/MS spectra of the undecaprenyl-phosphate sugars in E. coli strains.
Lipids extracted from E. coli strains were subjected to negative ion ESI/MS. Only regions of the spectra showing the [M-H]− ions of undecaprenyl-phosphate and undecaprenyl-phosphate sugars are shown. (A) Wild-type, polymyxin-sensitive, E. coli W3110. (B) polymyxin resistant E. coli W3110 pmrAc. (C) E. coli W3110 pmrAc ΔarnC. (D) E. coli W3110 pmrAc ΔarnD. The exact masses for C55P, C55P-Ara4N and C55P-Ara4FN are 846.665, 977.724 and 1005.719, respectively. The asterisk (*) denotes chloroform soluble CydH.
Figure 2:
Figure 2:. Crystal structure of Salmonella Typhimurium ArnD.
Top (A) and side (B) views of a cartoon representation of stArnD. Strands, helices, and coils in the NodB fold are colored light pink, pale cyan and light orange, respectively. The partially modeled 44 amino acid insertion is colored red and a C-terminal β-hairpin extension is colored blue. (C-D) Cartoon representation of the PgdA structure colored and oriented as in A and B. The bound metal ion is shown in red.
Figure 3:
Figure 3:. Conserved sequence motifs in ArnD vs Polysaccharide Deacetylases.
(A and D) Five motifs (MT1–5) previously described for the peptidoglycan deacetylase PgdA as a representative of polysaccharide deacetylases are shown in the active site of PgdA colored in light tints of pink (MT1), yellow (MT2), green (MT3), orange (MT4) and cyan (MT5). Bound Zn2+ ion and acetate are shown in red and black, respectively. (B and E) The corresponding but divergent motifs in ArnD are shown in bright hues of the same colors and a putative metal ion is modeled as a semitransparent sphere for reference. (C and F) Logos representations of sequence conservation in the five ArnD motifs, highlighted in boxes matching the color scheme in B-E.
Figure 4:
Figure 4:. ArnD membrane association models.
(A) stArnD with the NodB fold colored as in Fig. 2 with AlphaFold2-generated models for the 44 amino acid insertion. Three representative conformations for the insertion are shown colored in orange, yellow and green. (B) Membrane association models calculated using the PPM server for the three representative AlphaFold2 conformations shown in panel A. (C) Representation of the solvent accessible surface for the ArnD AlphaFold2 model with the membrane-interacting “finger” in the conformation depicted at the bottom of panel B, shown from the membrane plane. The surface is colored by the non-polar to polar residue ratio in a scale from 0.6 (polar) to 2.3 (non-polar). In all panels, a metal ion in the putative active site is shown as a red sphere.
Figure 5:
Figure 5:. ArnD is a metal-dependent lipid-phospho-L-Ara4FN deformylase.
(A) Thin layer chromatography separation of ArnD incubations with undecaprenyl-phospho-L-Ara4FN. UDP-Ara4FN was converted to C55P-Ara4FN by ArnC, and the product was incubated with purified ArnD for 10 minutes under the conditions indicated in each lane. (B) Thin layer chromatography separation of neryl-phosphate reactions with ArnC and ArnD. UDP-Ara4FN (lane 1) was incubated with neryl-phosphate plus (lane 2) or minus (lane 3) ArnC. The formylated amino-sugar is efficiently transferred by ArnC to the lipid to yield neryl-P-Ara4FN (C10P-Ara4FN). Lanes 4–8: The product of the ArnC reaction was incubated with purified ArnD for 10 minutes under the conditions indicated in each lane.
Figure 6:
Figure 6:. Proposed ArnD mechanism.
(A) Thin layer chromatography separations of reactions containing C10P-[14C]Ara4FN and wild type (WT) or mutant (D9N or H233Y) ArnD. Samples were taken at the indicated time points of the reaction. (B) Proposed reaction mechanism based on similarity to polysaccharide deacetylases (adapted from reference, copyright (2005) National Academy of Sciences, U.S.A.). A coordinated divalent metal is denoted as Me2+.
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