Discovery, characterization, and structural determination of a novel UDP‐2,3‐diacylglucosamine hydrolase

Abstract

Gram‐negative bacteria possess an asymmetric outer membrane in which the inner leaflet is composed primarily of phospholipids, while the outer leaflet contains mainly lipopolysaccharide (LPS). LPS forms a structural barrier that protects Gram‐negative bacteria from antibiotics and other environmental stressors. LPS is anchored to the outer membrane by Lipid A, a unique glucosamine‐based saccharolipid. Lipid A biosynthesis is required for viability and pathogenesis. While most lipid A biosynthetic genes are present in a single copy, one gene, lpxH, encoding a membrane‐associated specific UDP‐diacylglucosamine hydrolase, is absent in ~30% of Gram‐negative bacteria. We hypothesized that a transformational analogue of lpxH must exist in these organisms. We identified this gene, designated lpxI, in Caulobacter crescentus, and confirmed its ability to cover for a deficiency of lpxH in Eschserichia coli. LpxI lacks homology to any other known enzyme. We over‐expressed LpxI, purified it, and obtained protein crystals. We solved the x‐ray crystal structure of this peripheral membrane enzyme to a resolution of 2.7 angstroms using a single‐wavelength anomalous dispersion (SAD) dataset collected on Se‐Met derivatized protein. The structural data reveal two domains, each having a novel fold. Unexpectedly, “apo” LpxI co‐purified and co‐crystallized in a 1:1 stoichiometric ratio with its product, lipid X. These data provide an interesting example of lipid‐protein interaction, and set the stage for additional structural and mechanistic work. Support was provided by NIH grant GM5150 to C.R.H.R., Specialized Center grant of the Protein Structure Initiative U54 GM074929‐01 to R.M.S., and NSF GRF grant 2005029353 to L.E.M.