Crystal Structure of <i>Escherichia coli</i> ArnA (PmrI) Decarboxylase Domain. A Key Enzyme for Lipid A Modification with 4-Amino-4-deoxy-l-arabinose and Polymyxin Resistance<sup>†</sup><sup>,</sup><sup>‡</sup>
2004-10-26T00:00:00Z (GMT) by
Gram-negative bacteria including <i>Escherichia coli</i>, <i>Salmonella typhimurium</i>, and <i>Pseudomonas</i> <i>aeruginosa</i> 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<sup>+</sup>-dependent decarboxylation of UDP-glucuronic acid to UDP-4-keto-arabinose and (ii) the <i>N</i>-10-formyltetrahydrofolate-dependent formylation of UDP-4-amino-4-deoxy-l-arabinose. We show that the NAD<sup>+</sup>-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 <i>E. coli</i> 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<sup>+</sup> and UDP-glucuronic acid and the involvement of residues T<sub>432</sub>, Y<sub>463</sub>, K<sub>467</sub>, R<sub>619</sub>, and S<sub>433</sub> in the mechanism of NAD<sup>+</sup>-dependent oxidation of the 4‘ ‘-OH of the UDP-glucuronic acid and decarboxylation of the UDP-4-keto-glucuronic acid intermediate.