title: Structure and mechanism of ArnA: conformational change implies ordered dehydrogenase mechanism in key enzyme for polymyxin resistance.
abstract: 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-GlcA), and a transformylase domain that formylates UDP-Ara4N. Here, we describe the crystal structure of the full-length bifunctional ArnA with UDP-GlcA and ATP bound to the dehydrogenase domain. Binding of UDP-GlcA triggers a 17 A 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 R619 and S433 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado 80309, USA.
authors.forename: Petia Z
authors.initials: PZ
authors.lastname: Gatzeva-Topalova
authors.forename: Andrew P
authors.initials: AP
authors.lastname: May
authors.forename: Marcelo C
authors.initials: MC
authors.lastname: Sousa
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000606:Amino Sugars / Q000737:chemistry* / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D019281:Dimerization; D004352:Drug Resistance, Microbial; D006860:Hydrogen Bonding; D019877:Hydroxymethyl and Formyl Transferases / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D010084:Oxidation-Reduction; D011113:Polymyxins / Q000494:pharmacology*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013059:Spectrum Analysis, Raman; D013379:Substrate Specificity; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D014535:Uridine Diphosphate Glucuronic Acid / Q000737:chemistry / Q000378:metabolism
chemical_list: D000606:Amino Sugars; D000900:Anti-Bacterial Agents; D008024:Ligands; D011113:Polymyxins; D014535:Uridine Diphosphate Glucuronic Acid; C040134:4-amino-4-deoxyarabinose; D000255:Adenosine Triphosphate; D014533:Uridine Diphosphate Glucose Dehydrogenase; D019877:Hydroxymethyl and Formyl Transferases; C021561:methionyl-tRNA formyltransferase