Structure and Function of Both Domains of ArnA, a Dual Function Decarboxylase and a Formyltransferase, Involved in 4-Amino-4-deoxy-L-arabinose Biosynthesis

James H Naismith,Steven D Breazeale,Christian R.H Raetz,Gareth J Williams

doi:10.1074/jbc.m501534200

James H Naismith, Steven D Breazeale + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m501534200

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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.

Highlights

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
To synthesize UDP-LAra4FN, the N-terminal formyltransferase domain of ArnA catalyzes the transfer of a formyl group from N-10-fTHF to the 4Ј -amine of UDP-L-Ara4N
A similar analysis was performed by incubating purified ArnB with the N-terminal formyltransferase domain and the C-terminal decarboxylase domain of ArnA or the full-length ArnA protein

Summary

EXPERIMENTAL PROCEDURES

Overexpression of Native and Selenomethionine Decarboxylase and Formyltransferase Domains of ArnA—Full-length protein was cloned and expressed but failed to yield satisfactory crystals. Association of Domains with Each Other and with ArnB Association of Domains with Each Other and with ArnB—To investigate whether the isolated domains interact with each other, the purified domains, at ϳ3 mg mlϪ1 (ϳ0.075 mM), were mixed and incubated for 1 h at room temperature in buffer containing 50 mM HEPES, pH 7.5, 250 mM NaCl. The mixture was applied to an S200 gel filtration column, and the elution of proteins was followed by A280. Crystallization and Data Collection of Native and Se-Met Decarboxylase Protein—Good quality crystals were obtained with a precipitant solution of 3.2 M NaCl, 0.1 M Bistris, pH 5.2, using a drop containing 4 ␮l of protein and 4 ␮l of precipitant equilibrated against a reservoir of 100 ␮l of precipitant. Se-Met crystals were used to collect data to 3.35 Å on a MAR-CCD detector, at three wavelengths around the selenium absorption edge, on beamline BM14 at the European Synchrotron Radiation Facility. Structure Determination and Refinement of the Decarboxylase and Formyltransferase Proteins—SOLVE [32] located four selenomethionines for the decarboxylase data and eight selenomethionines for the Crystal Structure of ArnA

ArnA formyltransferase domain

RESULTS

Apparent Km mM

DISCUSSION