Structural biology of membrane-intrinsic beta-barrel enzymes: sentinels of the bacterial outer membrane

Abstract

The outer membranes of Gram-negative bacteria are replete with integral membrane proteins that exhibit antiparallel beta-barrel structures, but very few of these proteins function as enzymes. In Escherichia coli, only three beta-barrel enzymes are known to exist in the outer membrane; these are the phospholipase OMPLA, the protease OmpT, and the phospholipidColon, two colonslipid A palmitoyltransferase PagP, all of which have been characterized at the structural level. Structural details have also emerged for the outer membrane beta-barrel enzyme PagL, a lipid A 3-O-deacylase from Pseudomonas aeruginosa. Lipid A can be further modified in the outer membrane by two beta-barrel enzymes of unknown structure; namely, the Salmonella enterica 3'-acyloxyacyl hydrolase LpxR, and the Rhizobium leguminosarum oxidase LpxQ, which employs O(2) to convert the proximal glucosamine unit of lipid A into 2-aminogluconate. Structural biology now indicates how beta-barrel enzymes can function as sentinels that remain dormant when the outer membrane permeability barrier is intact. Host immune defenses and antibiotics that perturb this barrier can directly trigger beta-barrel enzymes in the outer membrane. The ensuing adaptive responses occur instantaneously and rapidly outpace other signal transduction mechanisms that similarly function to restore the outer membrane permeability barrier.

Fig. 1

Molecular organization of the Escherichia coli cell envelope. The outer membrane is an asymmetric bilayer with an inner leaflet of glycerophospholipids (GPL), and an outer leaflet of lipopolysaccharide, which can be divided into the lipid A, core oligosaccharide, and O-antigen (O_Ag) polysaccharide regions. The membrane-intrinsic proteins of the outer membrane are transmembrane β-barrels, while lipoproteins, anchored to the outer membrane inner leaflet, can provide a link with the murein exoskeleton. The energy-transducing inner membrane is a glycerophospholipid bilayer that supports the proton motive force and contains transmembrane α-helical proteins. The periplasmic space is the region between the inner and outer membranes and contains numerous globular proteins.

Fig. 2

Outer membrane transformations of glycerophospholipids and lipid A. PagP catalyzes transfer of a palmitate chain (C_16:0) from the sn-1 position of phosphatidylethanolamine (PtdEtn) to lipid A (endotoxin). Escherichia coli lipid A is a β-1′,6-linked disaccharide of glucosamine that is acylated with R-3-hydroxymyristate chains at the 2, 3, 2′, and 3′ positions, and phosphorylated at the 1 and 4′ positions. Acyloxyacyl linkages with laurate and myristate chains at the 2′ and 3′-positions, respectively, provide the constitutive hexa-acylated lipid A, which is a potent endotoxin. A regulated proportion of lipid A in E. coli contains a palmitate chain in acyloxyacyl linkage at position 2, which yields a hepta-acylated molecule that both is attenuated as an endotoxin and provides resistance to cationic antimicrobial peptides. PagL makes similar contributions by removing the R-3-hydroxymyristate chain at position 3 from either hexa-acyl or hepta-acyl lipid A. PagP can acylate the cytoplasmic monosaccharide lipid A precursor lipid X to produce lipid Y in vitro, and PagL can similarly deacylate lipids X and Y in vitro. The sn-1-lysophospholipid byproduct of the PagP reaction can be generated directly by the phospholipase activity of OMPLA when Ca²⁺ ions are available. OMPLA also displays lysophospholipase activity in vitro, which can release the sn-3-glycerophosphoethanolamine (sn-3-GroPEtn) polar head group from the remaining unsaturated fatty acyl chain (C_18:1).

Fig. 3

Structures of outer membrane β-barrel enzymes. (A) The outer membrane phospholipase OMPLA from E. coli with bound Ca²⁺ (purple) and hexadecanesulfonyl group (pdb: 1QD6). (B) The outer membrane protease OmpT from E. coli (pdb: 1I78); coordinates with bound lipooligosaccharide provided by Piet Gros and Lucy Rutten (Utrecht University). (C) The phospholipid::lipid A palmitoyltransferase PagP from E. coli with bound lauroyldimethylamine-N-oxide (pdb: 1THQ); coordinates with L1 loop introduced between β-strands A and B provided by Chris Neale and Régis Pomès (University of Toronto). (D) The outer membrane lipid A 3-O-deacylase from Pseudomonas aeruginosa (pdb 2ERV); coordinates with bound lipid X provided by Jan Tommassen and Lucy Rutten (Utrecht University).