Assignment of 1H, 13C and 15N backbone resonances of Escherichia coli LpxC bound to L-161,240

Abstract

The UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase LpxC catalyzes the committed reaction of lipid A biosynthesis, an essential pathway in Gram-negative bacteria. We report the backbone resonance assignments of the 34 kDa LpxC from Escherichia coli in complex with the antibiotic L-161,240 using multidimensional, multinuclear NMR experiments. The (1)H chemical shifts of complexed L-161,240 are also determined.

Fig. 1

¹⁵N-TROSY-HSQC spectrum of a the E. coli LpxC—L-161,240 complex at 800 MHz. Many backbone assignments were obtained from TROSY-based triple-resonance experiments including a TROSY-HNCA experiment (b). Remaining resonances were identified using a ¹⁵N-separated NOESY-TROSY-HSQC experiment (c). Connectivity in b is displayed with a thick red line. Amide self peaks (diagonals) in c are shown with a colored “+” symbol, and related cross peaks in adjacent strips are connected with a thick line of the same color as the marker. The amino acid identities are shown at the top of the figure, and the corresponding ¹H and ¹⁵N chemical shifts are at the bottom of each strip

Fig. 2

Secondary structure from the reported A. aeolicus LpxC (Coggins et al. 2003) and P. aeruginosa LpxC (Mochalkin et al. 2008) structures are compared with the predicted secondary structure of E. coli LpxC. An additional β-strand observed in P. aeruginosa LpxC and predicted for E. coli LpxC is marked at position 1. Gaps in the structural alignment are indicated with a “−”. Unobservable (Pro, N-terminal Met) or unassigned amide resonances of E. coli LpxC are indicated with red rectangles. The numbers correspond to the E. coli LpxC amino acid sequence