Structure and mechanism of ArnA: Conformational change implies ordered dehydrogenase mechanism in key enzyme for polymyxin resistance

The modification of lipid A with 4-amino-4-deoxy-L-arabinose (Ara4N) allows gram-negative bacteria to resist the antimicrobial activity of cationic antimicrobial peptides and antibiotics such as polymyxin. ArnA is the first enzyme specific to the lipid A-Ara4N pathway. It contains two functionally and physically separable domains: a dehydrogenase domain (ArnA_DH) catalyzing the NAD(+)-dependent oxidative decarboxylation of UDP-Glucuronic acid (UDP-GIcA), and a transformylase domain that formylates UDP-Ara4N. Here, we describe the crystal structure of the full-length bifunctional ArnA with UDP-GIcA and ATP bound to the dehydrogenase domain. Binding of UDP-GIcA triggers a 17 angstrom conformational change in ArnA_DH that opens the NAD(+) binding site while trapping UDP-GlcA. We propose an ordered mechanism of substrate binding and product release. Mutation of residues R-619 and S-433 demonstrates their importance in catalysis and suggests that R619 functions as a general acid in catalysis. The proposed mechanism for ArnA_DH has important implications for the design of selective inhibitors.