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.
Publication types
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Comparative Study
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adenosine Triphosphate / metabolism
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Amino Sugars / chemistry*
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Amino Sugars / metabolism
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Anti-Bacterial Agents / pharmacology*
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Binding Sites
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Catalysis
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Crystallography, X-Ray
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Dimerization
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Drug Resistance, Microbial*
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Hydrogen Bonding
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Hydroxymethyl and Formyl Transferases / chemistry
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Hydroxymethyl and Formyl Transferases / metabolism
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Ligands
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Models, Molecular
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Molecular Conformation
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Mutation
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Oxidation-Reduction
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Polymyxins / pharmacology*
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Protein Binding
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Protein Structure, Secondary
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Protein Structure, Tertiary
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Spectrum Analysis, Raman
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Substrate Specificity
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Uridine Diphosphate Glucose Dehydrogenase / chemistry*
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Uridine Diphosphate Glucose Dehydrogenase / genetics
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Uridine Diphosphate Glucuronic Acid / chemistry
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Uridine Diphosphate Glucuronic Acid / metabolism
Substances
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Amino Sugars
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Anti-Bacterial Agents
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Ligands
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Polymyxins
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Uridine Diphosphate Glucuronic Acid
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4-amino-4-deoxyarabinose
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Adenosine Triphosphate
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Uridine Diphosphate Glucose Dehydrogenase
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Hydroxymethyl and Formyl Transferases
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methionyl-tRNA formyltransferase
Associated data
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PDB/1Z0R
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PDB/1Z73
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PDB/1Z74
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PDB/1Z75
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PDB/1Z7B
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PDB/1Z7E
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PDB/R619E
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PDB/R619M
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PDB/R619Y
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PDB/S433A