Structure and function of both domains of ArnA, a dual function decarboxylase and a formyltransferase, involved in 4-amino-4-deoxy-L-arabinose biosynthesis

Abstract

Modification of the lipid A moiety of lipopolysaccharide by the addition of the sugar 4-amino-4-deoxy-L-arabinose (L-Ara4N) is a strategy adopted by pathogenic Gram-negative bacteria to evade cationic antimicrobial peptides produced by the innate immune system. L-Ara4N biosynthesis is therefore a potential anti-infective target, because inhibiting its synthesis would render certain pathogens more sensitive to the immune system. The bifunctional enzyme ArnA, which is required for L-Ara4N biosynthesis, catalyzes the NAD(+)-dependent oxidative decarboxylation of UDP-glucuronic acid to generate a UDP-4'-keto-pentose sugar and also catalyzes transfer of a formyl group from N-10-formyltetrahydrofolate to the 4'-amine of UDP-L-Ara4N. We now report the crystal structure of the N-terminal formyltransferase domain in a complex with uridine monophosphate and N-5-formyltetrahydrofolate. Using this structure, we identify the active site of formyltransfer in ArnA, including the key catalytic residues Asn(102), His(104), and Asp(140). Additionally, we have shown that residues Ser(433) and Glu(434) of the decarboxylase domain are required for the oxidative decarboxylation of UDP-GlcUA. An E434Q mutant is inactive, suggesting that chemical rather than steric properties of this residue are crucial in the decarboxylation reaction. Our data suggest that the decarboxylase domain catalyzes both hydride abstraction (oxidation) from the C-4' position and the subsequent decarboxylation.

Figure 1

The assembly process for the modification of LPS by L-Ara4N (22,24).

Figure 2

the structure of ArnA 2a The structure of the formyltransferase domain. The protein is represented as ribbons and the secondary structure elements are numbered as the text. A loop (residues N35-A40) that is missing in the structure is labeled *. The two subdomains are visible, the N-terminal subdomain is at the top of the figure and extends to the uridine ring, the C-terminal subdomain sits below the ring. The dimer found in the crystal is shown. The decarboxylase domain is attached to the C-terminus (labeled C). 2b The structure of the decarboxylase domain. The protein is represented as ribbons and the secondary structure elements are numbered. A loop (residues V604-D615) that is missing in the structure is labeled *. The monomeric unit is shown. The N-terminus of this domain (labeled N) is attached to the formyltransferase domain. 2c The unbiased Fo-Fc electron density map for N-5-fTHF found in the formyltransferase domain. The molecules are shown in stick with atoms colored; carbon yellow, nitrogen blue, oxygen red and phosphorous purple. The map is contoured at 3σ (0.22eÅ⁻³) 2d The unbiased Fo-Fc electron density map for UMP found in the formyltransferase domain. Contouring and atomic color are the same as Figure 2c.

Figure 2

the structure of ArnA 2a The structure of the formyltransferase domain. The protein is represented as ribbons and the secondary structure elements are numbered as the text. A loop (residues N35-A40) that is missing in the structure is labeled *. The two subdomains are visible, the N-terminal subdomain is at the top of the figure and extends to the uridine ring, the C-terminal subdomain sits below the ring. The dimer found in the crystal is shown. The decarboxylase domain is attached to the C-terminus (labeled C). 2b The structure of the decarboxylase domain. The protein is represented as ribbons and the secondary structure elements are numbered. A loop (residues V604-D615) that is missing in the structure is labeled *. The monomeric unit is shown. The N-terminus of this domain (labeled N) is attached to the formyltransferase domain. 2c The unbiased Fo-Fc electron density map for N-5-fTHF found in the formyltransferase domain. The molecules are shown in stick with atoms colored; carbon yellow, nitrogen blue, oxygen red and phosphorous purple. The map is contoured at 3σ (0.22eÅ⁻³) 2d The unbiased Fo-Fc electron density map for UMP found in the formyltransferase domain. Contouring and atomic color are the same as Figure 2c.

Figure 2

the structure of ArnA 2a The structure of the formyltransferase domain. The protein is represented as ribbons and the secondary structure elements are numbered as the text. A loop (residues N35-A40) that is missing in the structure is labeled *. The two subdomains are visible, the N-terminal subdomain is at the top of the figure and extends to the uridine ring, the C-terminal subdomain sits below the ring. The dimer found in the crystal is shown. The decarboxylase domain is attached to the C-terminus (labeled C). 2b The structure of the decarboxylase domain. The protein is represented as ribbons and the secondary structure elements are numbered. A loop (residues V604-D615) that is missing in the structure is labeled *. The monomeric unit is shown. The N-terminus of this domain (labeled N) is attached to the formyltransferase domain. 2c The unbiased Fo-Fc electron density map for N-5-fTHF found in the formyltransferase domain. The molecules are shown in stick with atoms colored; carbon yellow, nitrogen blue, oxygen red and phosphorous purple. The map is contoured at 3σ (0.22eÅ⁻³) 2d The unbiased Fo-Fc electron density map for UMP found in the formyltransferase domain. Contouring and atomic color are the same as Figure 2c.

Figure 2

the structure of ArnA 2a The structure of the formyltransferase domain. The protein is represented as ribbons and the secondary structure elements are numbered as the text. A loop (residues N35-A40) that is missing in the structure is labeled *. The two subdomains are visible, the N-terminal subdomain is at the top of the figure and extends to the uridine ring, the C-terminal subdomain sits below the ring. The dimer found in the crystal is shown. The decarboxylase domain is attached to the C-terminus (labeled C). 2b The structure of the decarboxylase domain. The protein is represented as ribbons and the secondary structure elements are numbered. A loop (residues V604-D615) that is missing in the structure is labeled *. The monomeric unit is shown. The N-terminus of this domain (labeled N) is attached to the formyltransferase domain. 2c The unbiased Fo-Fc electron density map for N-5-fTHF found in the formyltransferase domain. The molecules are shown in stick with atoms colored; carbon yellow, nitrogen blue, oxygen red and phosphorous purple. The map is contoured at 3σ (0.22eÅ⁻³) 2d The unbiased Fo-Fc electron density map for UMP found in the formyltransferase domain. Contouring and atomic color are the same as Figure 2c.

Figure 3

The active sites of ArnA 3a The active site of formyl transferase. The key catalytic residues are shown (*) as are the two substrate mimics (UMP and N-5-fTHF) located experimentally and those residues which recognize them. The atomic color scheme is the same as Figure 2c 3b The active site of the decarboxylase enzyme. We have modeled in the UDP-GlcUA substrate to generate a conceptual model. The model is based on a superposition of the RmlB substrate complex (42). The catalytic triad residues are shown as are the residues we predict to be important in the decarboxylation step (S433 and E434). These residues superimpose with D135 and E136 of RmlB which are responsible for catalysis of the dehydration step in RmlB transformation (42). The atomic color scheme is the same as Figure 2c.

Figure 3

The active sites of ArnA 3a The active site of formyl transferase. The key catalytic residues are shown (*) as are the two substrate mimics (UMP and N-5-fTHF) located experimentally and those residues which recognize them. The atomic color scheme is the same as Figure 2c 3b The active site of the decarboxylase enzyme. We have modeled in the UDP-GlcUA substrate to generate a conceptual model. The model is based on a superposition of the RmlB substrate complex (42). The catalytic triad residues are shown as are the residues we predict to be important in the decarboxylation step (S433 and E434). These residues superimpose with D135 and E136 of RmlB which are responsible for catalysis of the dehydration step in RmlB transformation (42). The atomic color scheme is the same as Figure 2c.

Figure 4

Sequence alignment of formyl transfer domains, numbering corresponds to full length ArnA. The key conserved catalytic residues are marked * as in Figure 3a, other conserved residues have a + underneath. ArnA residues that recognize N-5-fTHF and UMP are marked with ‡ and # respectively.

Figure 5

A plausible mechanism for the catalysis of the decarboxylation reaction.