Active-site architecture and catalytic mechanism of the lipid A deacylase LpxR of Salmonella typhimurium.
The lipid A portion of lipopolysaccharide, the major component of the outer leaflet of the outer membrane of gram-negative bacteria, is toxic to humans. Modification of lipid A by enzymes often reduces its toxicity. The outer-membrane protein LpxR from Salmonella typhimurium is a lipid A-modifying enzyme. It removes the 3'-acyloxyacyl moiety of the lipid A portion of lipopolysaccharide in a Ca(2+)-dependent manner. Here, we present the crystal structure of S. typhimurium LpxR, crystallized in the presence of zinc ions. The structure, a 12-stranded beta-barrel, reveals that the active site is located between the barrel wall and an alpha-helix formed by an extracellular loop. Based on site-directed mutagenesis and modeling of a substrate on the active site, we propose a catalytic mechanism similar to that of phospholipase A2, in which a Ca(2+) forms the oxyanion hole and a histidine activates a water molecule (or a cascade of two water molecules) that subsequently attacks the carbonyl oxygen of the scissile bond.
Rutten
Lucy
L
Laboratory of Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands.
Mannie
Jean-Paul B A
JP
Stead
Christopher M
CM
Raetz
Christian R H
CR
Reynolds
C Michael
CM
Bonvin
Alexandre M J J
AM
Tommassen
Jan P
JP
Egmond
Maarten R
MR
Trent
M Stephen
MS
Gros
Piet
P
D002118:Calcium; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006639:Histidine; D008050:Lipid A / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000378:metabolism; D014867:Water; D015032:Zinc
D008050:Lipid A; D014867:Water; D006639:Histidine; D002265:Carboxylic Ester Hydrolases; C513984:LpxR protein, Salmonella typhimurium; D015032:Zinc; D002118:Calcium