Crystal structure and activity of Francisella novicida UDP-N-acetylglucosamine acyltransferase

Abstract

The first step of lipid A biosynthesis in Escherichia coli (E. coli) is catalyzed by LpxA (EcLpxA), an acyltransferase selective for UDP-N-acetylglucosamine (UDP-GlcNAc) and R-3-hydroxymyristoyl-acyl carrier protein (3-OH-C-14-ACP), and is an essential step in majority of Gram-negative bacteria. Since the majority of lipid A species isolated from F. novicida contains 3-OH-C-16 or 3-OH-C-18 at its C3 and C30 positions, FnLpxA was thought to be selective for longer acyl chain (3-OH-C-16 and 3-OH-C-18) over short acyl chain (3-OH-C14, 3-OH-C-12, and 3-OH-C-10). Here we demonstrate that Francisella novicida (F. novicida) lpxA functionally complements an E. coli lpxA knockout mutant and efficiently transfers 3-OH-C14 as well as 3OH- C-16 in E. coli. Our results implicate that the acyl chain length of lipid A is determined by several factors including acyl chain selectivity of LpxA and downstream enzymes, as well as the composition of the acyl-ACP pool in vivo. We also report the crystal structure of F. novicida LpxA (FnLpxA) at 2.06 angstrom. The N-terminal parallel beta-helix (LbH) and C-terminal alpha-helical domain are similar to other reported structures of LpxAs. However, our structure indicates that the supposed ruler residues for hydrocarbon length, 171L in one monomer and 168H in the adjacent monomer in a functional trimer of FnLpxA, are located just 3.8 angstrom apart that renders not enough space for binding of 3-OH-C-12 or longer acyl chains. This implicates that FnLpxA may have an alternative hydrophobic pocket, or the acyl chain may bend while binding to FnLpxA. In addition, the FnLpxA structure suggests a potential inhibitor binding site for development of antibiotics. (C) 2016 Elsevier Inc. All rights reserved.