Structural Basis for the Recognition of Peptide RJPXD33 by Acyltransferases in Lipid A Biosynthesis

Jenkins, Ronald J.; Heslip, Kyle A.; Meagher, Jennifer L.; Stuckey, Jeanne A.; Dotson, Garry D.

doi:10.1074/jbc.m114.564278

Cited by 26 publications

(40 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Notably, the position of the tag coincides very well with the reported binding location of peptide inhibitors of EcLpxA, Peptide 920 [30] (Fig. 4C) and RJPXD33 [31] (Fig. 4D).…”

Section: Resultssupporting

confidence: 79%

See 1 more Smart Citation

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

Joo

Chung

2016

Biochemical and Biophysical Research Communications

View full text Add to dashboard Cite

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-C14-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-C16 or 3-OH-C18 at its C3 and C3' positions, FnLpxA was thought to be selective for longer acyl chain (3-OH-C16 and 3-OH-C18) over short acyl chain (3-OH-C14, 3-OH-C12, and 3-OH-C10). 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 3-OH-C16 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 Å. The N-terminal parallel beta-helix (LβH) 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 Å apart that renders not enough space for binding of 3-OH-C12 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.

show abstract

“…Notably, the position of the tag coincides very well with the reported binding location of peptide inhibitors of EcLpxA, Peptide 920 [30] (Fig. 4C) and RJPXD33 [31] (Fig. 4D).…”

Section: Resultssupporting

confidence: 79%

“…(PDB id: 2AQ9 [30]). D. RJPXD33, another peptide inhibitor (salmon sticks) of EcLpxA, is located at the interface of two adjacent monomers of EcLpxA in the functional trimer (PDB id: 4J09 [31]). In the inset, the FnLpxA trimer and EcLpxE trimers are overlaid and the binding sites of individual structures are enlarged (boxed region) in Fig.…”

Section: Figurementioning

confidence: 99%

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

Joo

Chung

2016

Biochemical and Biophysical Research Communications

View full text Add to dashboard Cite

show abstract

“…Indeed, the optimization of LpxC inhibitors has been an ongoing effort in antimicrobial research for over 2 decades, which has yielded compounds with impressive antibacterial activity against Gram-negative organisms such as Pseudomonas aeruginosa (1,(3)(4)(5)(6)(7)(8)(9). The identification of RJPXD33, an antimicrobial peptide that inhibits both Escherichia coli LpxA and LpxD, and a recently identified LpxH inhibitor suggests that other LPS biosynthetic steps could also be successfully targeted (10)(11)(12). POL7001, a peptidomimetic antibiotic that inhibits LptD, the final essential step of the LPS transport (Lpt) system, has potent and specific antibacterial activity against P. aeruginosa, which, importantly, indicates that the LPS transport and OM assembly machinery may be attractive targets for antibacterial discovery (13)(14)(15).…”

mentioning

confidence: 99%

Characterization of an Acinetobacter baumannii lptD Deletion Strain: Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis

et al. 2016

View full text Add to dashboard Cite

Lipid A on the Gram-negative outer membrane (OM) is synthesized in the cytoplasm by the Lpx pathway and translocated to the OM by the Lpt pathway. Some Acinetobacter baumannii strains can tolerate the complete loss of lipopolysaccharide (LPS) resulting from the inactivation of early LPS pathway genes such as lpxC. Here, we characterized a mutant deleted for lptD, which encodes an OM protein that mediates the final translocation of fully synthesized LPS to the OM. Cells lacking lptD had a growth defect comparable to that of an lpxC deletion mutant under the growth conditions tested but were more sensitive to hydrophobic antibiotics, revealing a more significant impact on cell permeability from impaired LPS translocation than from the loss of LPS synthesis. Consistent with this, ATP leakage and N-phenyl-1-naphthylamine (NPN) fluorescence assays indicated a more severe impact of lptD deletion than of lpxC deletion on inner and outer membrane permeability, respectively. Targeted In most Gram-negative bacteria, many of the proteins responsible for lipopolysaccharide (LPS) synthesis and transport are essential, making them potential targets for antibacterial drug development. In particular, the nine conserved Lpx enzymes are considered promising for the development of novel antibiotics, since (3-deoxy-D-manno-oct-2-ulosonic acid) 2 -lipid A (Kdo 2 -lipid A) is the minimal LPS structure that supports a functional outer membrane (OM) and cell viability for most Gram-negative bacteria (1, 2). Indeed, the optimization of LpxC inhibitors has been an ongoing effort in antimicrobial research for over 2 decades, which has yielded compounds with impressive antibacterial activity against Gram-negative organisms such as Pseudomonas aeruginosa (1,(3)(4)(5)(6)(7)(8)(9). The identification of RJPXD33, an antimicrobial peptide that inhibits both Escherichia coli LpxA and LpxD, and a recently identified LpxH inhibitor suggests that other LPS biosynthetic steps could also be successfully targeted (10-12). POL7001, a peptidomimetic antibiotic that inhibits LptD, the final essential step of the LPS transport (Lpt) system, has potent and specific antibacterial activity against P. aeruginosa, which, importantly, indicates that the LPS transport and OM assembly machinery may be attractive targets for antibacterial discovery (13)(14)(15).Acinetobacter baumannii is an emerging opportunistic bacterial pathogen of increasing concern due to multidrug resistance (16). A. baumannii is noted for its ability to develop resistance against most conventional antibiotics through mechanisms such as the upregulation of efflux pumps and horizontal transfer of resistance genes (17)(18)(19). Because of this, clinical resistance is becoming a serious issue for this organism (as well as for other Gram-negative pathogens), often necessitating the use of antibiotics of last resort, such as polymyxins (19,20). Understanding resistance to polymyxins, which are cationic and utilize LPS to gain access to cells, has therefore become a focus of renewed in-

show abstract

“…The structure of RJPXD33 was captured in complex with E. coli LpxA ( Fig. 2F ), revealing an extended conformation that is distinct from the hairpin structure of peptide 920 [30]. The peptide backbone of RJPXD33 runs parallel with the acyl chain of the UDP-3- O -acyl-GlcNAc molecule, with its amino acid sidechains reaching out to the space occupied by the acyl chain and the UDP-GlcNAc moiety ( Fig.…”

Section: Lpxamentioning

confidence: 99%

Structure, inhibition, and regulation of essential lipid A enzymes

Zhou

Zhao

2017

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

View full text Add to dashboard Cite

The Raetz pathway of lipid A biosynthesis plays a vital role in the survival and fitness of Gram-negative bacteria. Research efforts in the past three decades have identified individual enzymes of the pathway and have provided a mechanistic understanding of the action and regulation of these enzymes at the molecular level. This article reviews the discovery, biochemical and structural characterization, and regulation of the essential lipid A enzymes, as well as continued efforts to develop novel antibiotics against Gram-negative pathogens by targeting lipid A biosynthesis.

show abstract

Structural Basis for the Recognition of Peptide RJPXD33 by Acyltransferases in Lipid A Biosynthesis

Cited by 26 publications

References 34 publications

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

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

Characterization of an Acinetobacter baumannii lptD Deletion Strain: Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis

Structure, inhibition, and regulation of essential lipid A enzymes

Contact Info

Product

Resources

About