Crystal structure of Escherichia coli ArnA (PmrI) decarboxylase domain. A key enzyme for lipid A modification with 4-amino-4-deoxy-L-arabinose and polymyxin resistance

Abstract

Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa can modify the structure of lipid A in their outer membrane with 4-amino-4-deoxy-l-arabinose (Ara4N). Such modification results in resistance to cationic antimicrobial peptides of the innate immune system and antibiotics such as polymyxin. ArnA is a key enzyme in the lipid A modification pathway, and its deletion abolishes both the Ara4N-lipid A modification and polymyxin resistance. ArnA is a bifunctional enzyme. It can catalyze (i) the NAD(+)-dependent decarboxylation of UDP-glucuronic acid to UDP-4-keto-arabinose and (ii) the N-10-formyltetrahydrofolate-dependent formylation of UDP-4-amino-4-deoxy-l-arabinose. We show that the NAD(+)-dependent decarboxylating activity is contained in the 360 amino acid C-terminal domain of ArnA. This domain is separable from the N-terminal fragment, and its activity is identical to that of the full-length enzyme. The crystal structure of the ArnA decarboxylase domain from E. coli is presented here. The structure confirms that the enzyme belongs to the short-chain dehydrogenase/reductase (SDR) family. On the basis of sequence and structure comparisons of the ArnA decarboxylase domain with other members of the short-chain dehydrogenase/reductase (SDR) family, we propose a binding model for NAD(+) and UDP-glucuronic acid and the involvement of residues T(432), Y(463), K(467), R(619), and S(433) in the mechanism of NAD(+)-dependent oxidation of the 4''-OH of the UDP-glucuronic acid and decarboxylation of the UDP-4-keto-glucuronic acid intermediate.

FIGURE 1

Proposed pathway for the biosynthesis of UDP-Ara4N. The pathway starts with Ugd/PmrE oxidizing UDP-Glc to UDP-GlcA. UDP-GlcA is then oxidized at position 4 by the C-terminal domain of ArnA to yield the UDP-4-keto glucuronic acid intermediate that is then decarboxylated to UDP-4-keto arabinose by the same enzyme. UDP-Ara4O is transaminated by ArnB yielding the novel sugar-nucleotide UDP-4-amino-4-deoxy-arabinose (UDP-Ara4N). The N-terminal domain of ArnA can formylate UDP-Ara4N and has been proposed to help displace the reaction catalyzed by ArnB toward UDP-Ara4N synthesis and generate a transiently formylated product (21).

FIGURE 2

Schematic representation of the reactions catalyzed by some SDR enzymes. (A) ArnA decarboxylase domain. (B) UDP-Galactose epimerase. (C) UDP-Glucuronic acid decarboxylase. (D) dTDP glucose-4,6-dehydratase.

FIGURE 3

Decarboxylase activity of full-length ArnA and its C-terminal domain. (A) Plots of initial velocity versus substrate concentration for full-length ArnA (○) and ArnA C-terminal domain (●). The enzyme activity was measured by monitoring the formation of NADH. (B) Detection of the reaction product UDP-Ara4O by thin-layer chromatography. Lanes 1 and 2, Full-length ArnA; lanes 3 and 4, ArnA C-terminal domain. No formation of UDP-Ara4O is observed in the absence of NAD⁺ in the reaction mixture.

FIGURE 4

Overall structure of the ArnA decarboxylase domain. The N-terminal subdomain (in gold) is formed by residues R_315-R₅₁₀ and R₅₄₁-G₅₆₆. It adopts a modified version of the classic Rossmann fold in that an α helix and a β strand are donated by the C-terminal subdomain (shown in dark blue). The C-terminal subdomain (in blue) is formed by residues A₅₁₁-I₅₄₀ and N_567-T₆₅₆. All molecular diagrams were prepared with Molscript (52) and rendered with Raster 3D (53).

FIGURE 5

(A) Crystal structure of the E. coli ArnA decarboxylase domain with substrates modeled in the active site. (B) Crystal structure of E. coli UDP-galactose 4-epimerase with its substrates bound in the active site (PDB ID: 1A9Y). The two loops highlighted in magenta and gold in both proteins reveal structural differences likely to be important in substrate binding.

FIGURE 6

Conformational differences between the ArnA decarboxylase domain and UDP-galactose epimerase in the NAD⁺-binding loop that contacts the adenine. The loop is highlighted in gold and shows relevant residues in ecGALE hydrogen bonding with the adenine base. There is a four amino acid residue deletion in this loop in ArnA, and its conformation would prevent contact with NAD⁺.

FIGURE 7

Sequence alignment of members of the SDR family and secondary-structure assignment of ArnA decarboxylase. α helices are shown as green cylinders and β strands, as red arrows. The proteins are E. coli UDP-galactose 4-epimerase (UDP-Gal epimerase); E. coli dTDP-glucose-4,6-dehydratase (dTDP-Glc dehydratase); E. coli ADP-glycero-mannoheptose 6-epimerase (AGM epimerase); S. thyphi CDP-tyvelose 2-epimerase (CDP-tyvelose epimerase); A. thaliana UDP-glucose sulfotransferase also known as UDP-sulfoquinovose synthase (UDP-Glc sulfotransferase); Homo sapiens UDP-glucuronic acid decarboxylase (UDP-GlcA decarboxylase); and E. coli ArnA C-terminal (decarboxylase) domain (ArnA C terminus). The catalytic residues S/T, Y, and K, and the NAD-binding glycine-rich motif GXXGXXG are shaded in red. Other strictly conserved residues are shaded in dark orange, while less conserved residues are shaded in light orange and yellow. The serine and arginine residues that we propose to be involved in decarboxylation are shaded in green. The corresponding residues in other proteins are shaded in blue.

FIGURE 8

Arrangement of R₆₁₉ and S₄₃₃ in the vicinity of the UDPGlcA carboxylate. The strict conservation of these residues in decarboxylases reveals their potential importance for the decarboxylation reaction.