Biophysical and biochemical characterization of ArnA: A required enzyme in the polymyxin resistance pathway

Gram-negative bacteria can modify the structure of lipid A in their outer membrane with the positively charged 4-amino-4-deoxy-L-arabinose (Ara4N). Such modification results in resistance to cationic antimicrobial peptides of the innate immune system, and certain 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. A clear understanding of the ArnA structure and mechanism is crucial for the design of selective inhibitors of the pathway. Such inhibitors may prove particularly useful in treating chronic infections like those caused by bacteria in the lungs of Cystic Fibrosis (CF) patients.;ArnA is a bifunctional enzyme. It can catalyze (i) the oxidative decarboxylation of UDP-glucuronic acid (UDP-GlcA) to UDP-4-ketopentose (UDP-Ara4O) and (ii) the N-10-formyltetrahydrofolate dependent formylation of UDP-4-amino-4-deoxy-L-arabinose (UDP-Ara4N). We show that the transformylation activity is contained, exclusively, in the 300 amino acid N-terminal domain of ArnA, and the NAD+-dependent decarboxylating activity is contained in the 360 amino acid C-terminal domain of the enzyme. The crystal structures of the unliganded individual domains, as well as the structures of the liganded (with ATP/UDP-GlcA and UDP-GlcA alone) full-length ArnA from E. coli and S. thyphimurium are presented in this work.;A mechanism for the N-terminal transformylation reaction is proposed based on mutational and structural data. It is also proposed that the dehydrogenation reaction catalyzed by the C-terminus of ArnA follows an ordered mechanism in which UDP-GlcA is the first substrate to bind, inducing a large conformational change that opens the active site for NAD+. Sequence comparisons of the ArnA dehydrogenase domain with enzymes annotated as UDP-xylose synthases and other members of the Short Chain Dehydrogenase/Reductase (SDR) family allowed the identification of catalytic residues. Three residues---T 432, Y463, K467 are implicated in the oxidation of UDP-GlcA to UDP-4-keto glucuronic acid. Mutational studies confirm that two additional residues---R619 and S433 facilitate the decarboxylation of the UDP-4-keto glucuronic acid intermediate to yield the sugar nucleotide UDP-Ara4O. The above-mentioned results of this study have important implications for the design of selective inhibitors of ArnA.