Journal of Molecular Biology
The Structure of the Neisserial Lipooligosaccharide Phosphoethanolamine Transferase A (LptA) Required for Resistance to Polymyxin
Graphical Abstract
Introduction
Multidrug-resistant Gram-negative bacteria are a major cause of sepsis in intensive care units [1]. With the loss in utility of the major classes of antibiotics in multidrug-resistant Gram-negative bacteria, attention has returned to the use of polymyxins (colistin) for the treatment of sepsis [2]. Polymyxin is a cationic polypeptide ring with a lipophilic fatty acyl side chain. The amphipathic nature of the molecule allows it to bind to the bacterial surface and intercalate into the outer membrane. From this location, it diffuses across the periplasm and intercalates into the inner membrane, forming pores that eventually lead to bacterial cell lysis. Initial interactions with the bacterial surface rely on the electrostatic interaction between the positively charged polymyxin and the negative charge on the bacterial surface generated by the phosphate groups on the lipid A of lipopolysaccharide (LPS). LPS, which makes up to 70% of the outer leaflet of the outer membrane, consists of the lipid A moiety attached to glycan chain of variable length and composition. LPS is synthesized in the cytoplasm and flipped across the inner membrane into the periplasm by the ABC transporter, MsbA, before being transported to the outer membrane [3]. To prevent binding by polymyxin to lipid A, we modified the 1 and 4′ phosphates are modified with 4-amino-arabinose and phosphoethanolamine (PEA). The addition of PEA to lipid A by lipid A PEA transferases (LptAs) (see Fig. 1) is a major mechanism for resistance to polymyxin in Neisseria gonorrhoeae, N. meningitidis [7], and Acinetobacter [8], [9] since these species do not synthesize 4-amino-arabinose. The addition of 4-amino-arabinose to lipid A is commonly seen in Enterobacteriaceae and is regulated by two-component systems responding to pH, antimicrobial compounds, and metal ions [10].
Ethanolamine transferases are members of the YhjW/YjdB/YijP superfamily [11]. They consist of a hydrophobic transmembrane domain, which is located in the periplasmic membrane and is linked to a globular C-terminal sulfatase domain, which is presented on the periplasmic side of the membrane [12]. Other members of this family include the E. coli EptB (phosphoethanolamine transferase B), which catalyzes the transfer of PEA from phosphatidylethanolamine to the outer Kdo residue of LPS [13], PmrC (now renamed EptA), which catalyzes the transfer of PEA to the phosphate headgroups of lipid A in Salmonella enterica [14], and Lpt3 and Lpt6, which transfer PEA to positions O-3 and O-6 of the heptose residue II (HepII) of neisserial LPS, respectively [15], [16]. PptA (pilin phosphoethanolamine transferase), the enzyme responsible for catalyzing the transfer of PEA from phosphatidylethanolamine to neisserial Type IV pilin [12], [17], has also been found to be homologous to LptA and EptB and, based on a modeling study, the highest-quality models were derived from the structure of human arylsulfatases and N-acetylgalactosamine-4-sulfatase [12].
Currently, the catalytic mechanism of PEA transferases is unknown. In order to understand the molecular features of substrate binding and catalysis by this family of enzymes and to direct the design of inhibitors of the enzyme as potential novel antimicrobial agents in the treatment of Gram-negative bacterial infections, we have determined the three-dimensional structure of the soluble catalytic domain of LptA from Neisseria meningitidis by crystallographic methods.
Section snippets
sNMLptA is active in an aqueous assay but unable to add PEA to lipid A in Escherichia coli
A deletion construct of the enzyme where the predicted membrane anchor domain was removed giving only the soluble periplasmic domain of the protein was expressed from pCMK527 as sNMLptA. To determine whether this portion of the enzyme was active, we used a soluble chromogenic substrate para-nitrophenyl phosphoethanolamine (p-NPPE). Incubation of the purified, non-trypsin-treated sNMLptA with the chromogenic substrate, p-NPPE, resulted in the release of para-nitrophenolate ion giving a strong
Discussion
LptA is a member of the LPS PE transferase family, YhjW/YjdB/YijP, of which there are a variety of enzymes catalyzing the transfer of PEA to different centers of LPS. These enzymes make up a subfamily of the alkaline phosphatase superfamily [12], [13], [14], [15], [16], [17] and generally consist of a transmembrane domain attached to a globular domain facing the periplasmic space. Modeling studies of the pilin PEA transferase, PptA, were used to suggest putative metal-binding residues as
Protein expression, purification, and crystallization
The soluble domain of the enzyme without the membrane anchor portion (sNMLptA) was prepared as described from pCMK527 [18]. In order to determine the structure of the protein using the MAD phasing method, we expressed a SeMet mutant of the enzyme (sNMLptA-Se) by inhibiting the methionine biosynthesis pathway in E. coli through the addition of high concentrations of amino acids to block methionine biosynthesis and adding SeMet in place of methionine [30], [31], [29]. sNMLptA-Se protein was
Acknowledgements
The authors wish to thank the Australian Synchrotron (MX2 beamline), and Dr. Tom Caradoc-Davies, Dr. Rachel Williamson, and Dr. Nathan Cowieson for their assistance with remote data collection access. K.A.S. thanks the Australian Research Council for funding. The Centre for Microscopy, Characterisation & Analysis is thanked for assistance. This work was supported by a grant from the National Health and Medical Research Council of Australia (APP1003697) to A.V. and C.M.K.
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