8T0J

Salmonella Typhimurium ArnD

Summary for 8T0J

• Entry DOI: 10.2210/pdb8t0j/pdb
• Descriptor: Probable 4-deoxy-4-formamido-L-arabinose-phosphoundecaprenol deformylase ArnD (2 entities in total)
• Functional Keywords: polymyxin resistance, undecaprenyl-phospho-4-deoxy-4-formamido-arabinose deformylase, carbohydrate esterase, hydrolase
• Biological source: Salmonella enterica subsp. enterica serovar Typhimurium
• Total number of polymer chains: 1
• Total formula weight: 32951.66

Authors

Sousa, M.C.,Munoz-Escudero, D.,Lee, M. (deposition date: 2023-06-01, release date: 2023-10-18, Last modification date: 2023-11-01)

Primary citation

Munoz-Escudero, D.,Breazeale, S.D.,Lee, M.,Guan, Z.,Raetz, C.R.H.,Sousa, M.C.
Structure and Function of ArnD. A Deformylase Essential for Lipid A Modification with 4-Amino-4-deoxy-l-arabinose and Polymyxin Resistance.
Biochemistry, 62:2970-2981, 2023

PubMed 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 and 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 gene in an mutant that constitutively expresses the 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 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 Co or Mn, 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.

PubMed: 37782650
DOI: 10.1021/acs.biochem.3c00293

Experimental method

X-RAY DIFFRACTION (2.59 Å)