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Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria
Published on: September 16, 2013
Structure and Function of ArnD. A Deformylase Essential for Lipid A Modification with 4-Amino-4-deoxy-l-arabinose and
Daniel Muñoz-Escudero1, Steven D Breazeale2, Myeongseon Lee3
1Department of Molecular Cellular and Developmental Biology, University of Colorado Boulder, Boulder, Colorado 80309, United States.
Abstract:
Covalent modification of lipid A with 4-deoxy-4-amino-l-arabinose (Ara4N) mediates resistance to cationic antimicrobial peptides and polymyxin antibiotics in Gram-negative bacteria. The proteins required for Ara4N biosynthesis are encoded in the pmrE and arnBCADTEF loci, with ArnT ultimately transferring the amino sugar from undecaprenyl-phospho-4-deoxy-4-amino-l-arabinose (C55P-Ara4N) to lipid A. However, Ara4N is N-formylated prior to its transfer to undecaprenyl-phosphate by ArnC, requiring a deformylase activity downstream in the pathway to generate the final C55P-Ara4N donor. Here, we show that deletion of the arnD gene in an Escherichia coli mutant that constitutively expresses the arnBCADTEF operon leads to accumulation of the formylated ArnC product undecaprenyl-phospho-4-deoxy-4-formamido-l-arabinose (C55P-Ara4FN), suggesting that ArnD is the downstream deformylase. Purification of Salmonella typhimurium ArnD (stArnD) shows that it is membrane-associated. We present the crystal structure of stArnD revealing a NodB homology domain structure characteristic of the metal-dependent carbohydrate esterase family 4 (CE4). However, ArnD displays several distinct features: a 44 amino acid insertion, a C-terminal extension in the NodB fold, and sequence divergence in the five motifs that define the CE4 family, suggesting that ArnD represents a new family of carbohydrate esterases. The insertion is responsible for membrane association as its deletion results in a soluble ArnD variant. The active site retains a metal coordination H-H-D triad, and in the presence of Co2+ or Mn2+, purified stArnD efficiently deformylates C55P-Ara4FN confirming its role in Ara4N biosynthesis. Mutations D9N and H233Y completely inactivate stArnD implicating these two residues in a metal-assisted acid-base catalytic mechanism.
Insights
ArnD is identified as the enzyme responsible for removing a formyl group from lipid A precursors in Gram-negative bacteria. This discovery clarifies a key step in antibiotic resistance mechanisms and reveals a novel family of carbohydrate esterases.
Area of Science:
- Microbiology and Molecular Biology
- Biochemistry and Enzymology
- Structural Biology
Background:
- Gram-negative bacteria develop resistance to antimicrobial peptides and polymyxin antibiotics through covalent modification of lipid A with 4-deoxy-4-amino-l-arabinose (Ara4N).
- The biosynthesis pathway involves N-formylation of Ara4N by ArnC, necessitating a downstream deformylase to generate the active lipid A precursor (C55P-Ara4N).
Purpose of the Study:
- To identify the specific enzyme responsible for the deformylation step in the Ara4N biosynthesis pathway.
- To characterize the structure and function of the identified deformylase, ArnD.
Main Methods:
- Genetic analysis using an Escherichia coli mutant lacking the arnD gene to observe accumulation of formylated precursors.
- Purification and biochemical characterization of Salmonella typhimurium ArnD (stArnD).
- Determination of the crystal structure of stArnD and site-directed mutagenesis to probe active site residues.
Main Results:
- Deletion of arnD led to the accumulation of undecaprenyl-phospho-4-deoxy-4-formamido-l-arabinose (C55P-Ara4FN), confirming ArnD's role as the deformylase.
- The crystal structure of stArnD revealed a novel carbohydrate esterase family (CE4) with unique features, including a membrane-associating insertion.
- stArnD requires divalent metal ions (Co2+ or Mn2+) for activity and efficiently deformylates C55P-Ara4FN, with mutations D9N and H233Y inactivating the enzyme.
Conclusions:
- ArnD is the essential downstream deformylase in the Ara4N biosynthesis pathway, crucial for antibiotic resistance in Gram-negative bacteria.
- ArnD represents a novel family of membrane-associated, metal-dependent carbohydrate esterases with a unique structural architecture.
- Key active site residues D9 and H233 are critical for ArnD's catalytic mechanism, likely involving metal-assisted acid-base catalysis.
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