The Mechanism of Action of Lysobactin

Lee, Wonsik; Schaefer, Kaitlin; Qiao, Yuan; Srisuknimit, Veerasak; Steinmetz, Heinrich; Müller, Rolf; Kahne, Daniel; Walker, Suzanne

doi:10.1021/jacs.5b11807

Cited by 63 publications

(68 citation statements)

References 36 publications

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“…LI WTA was synthesized in six chemical steps and we converted this monosaccharide precursor enzymatically to the corresponding disaccharide, LII A WTA , using the UDP-ManNAc transferase TagA, UDP-GlcNAc, and the UDP-GlcNAc 2-epimerase, MnaA (Fig. 2a) 27,28 . A radiolabel was optionally incorporated using UDP-[ 14 C]-GlcNAc.…”

Section: Resultsmentioning

confidence: 99%

In vitro reconstitution demonstrates the cell wall ligase activity of LCP proteins

et al. 2017

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Sacculus is a peptidoglycan matrix that protects bacteria from osmotic lysis. In Gram-positive organisms, the sacculus is densely functionalized with glycopolymers important for survival, but how assembly occurs is not known. In Staphylococcus aureus, three LCP family members have been implicated in attaching the major glycopolymer wall teichoic acid (WTA) to peptidoglycan, but ligase activity has not been demonstrated for these or any other LCP proteins. Using WTA and peptidoglycan substrates produced chemoenzymatically, we show that all three proteins can transfer WTA precursors to nascent peptidoglycan, establishing that LCP proteins are peptidoglycan-glycopolymer ligases. Although all S. aureus LCP proteins have the capacity to attach WTA to PG, we show that their cellular functions are not redundant. Strains lacking lcpA have phenotypes similar to WTA null strains, indicating that this is the most important WTA ligase. This work provides a foundation for studying how LCP enzymes participate in cell wall assembly.

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Section: Resultsmentioning

confidence: 99%

In vitro reconstitution demonstrates the cell wall ligase activity of LCP proteins

et al. 2017

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“…The ladder of bands observed in the Sa PBP4 reaction is due to extensive crosslinking of 1 . 7 We attribute the small amount of cross-linking observed with Ef PBPX to the sterically unbiased glycine nucleophile of 1 , which can occasionally intercept activated stem peptides. These results supported our hypothesis that DUF1958-containing PBPs have transpeptidase activity.…”

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confidence: 96%

“…6c Unfortunately, it also crosslinks S. aureus Lipid II ( 1 ) extensively, precluding further use of the labeled material. 7 Here, we report a new family of low molecular weight PBPs that have transpeptidase activity. These enzymes can be used to label Gram-positive Lipid II variants, including S. aureus Lipid II.…”

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confidence: 97%

Identification of a Functionally Unique Family of Penicillin-Binding Proteins

et al. 2017

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Penicillin-binding proteins (PBPs) are enzymes involved in the assembly of the bacterial cell wall, a major target for antibiotics. These proteins are classified by mass into high molecular weight PBPs, which are transpeptidases that form peptidoglycan crosslinks, and low molecular weight PBPs, which are typically hydrolases. We report a functionally unique family of low molecular weight PBPs that act as transpeptidases rather than hydrolases, but they do not crosslink peptidoglycan. We show that these PBPs can exchange D-amino acids bearing chemical tags or affinity handles into peptidoglycan precursors, including Lipid II, enabling biochemical studies of proteins involved in cell wall assembly. We report that, in two organisms, the PBPs incorporate lysine into peptidoglycan and, further, that this linkage can occur via attack of the primary ε-amine side chain, an unprecedented reaction for a PBP.

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“…31,32 This behavior is due to the fact that blocking a late step in WTA biosynthesis depletes Lipid II, the peptidoglycan precursor, which is synthesized on the same undecaprenyl phosphate carrier lipid as the WTA precursor. 33,34 Therefore, it is possible to identify compounds that inhibit a late step in WTA biosynthesis by monitoring growth of a wildtype S. aureus strain and a Δ tarO mutant in which the first gene in the pathway has been deleted. From a screen of ~55,000 compounds, we identified three compounds that inhibited growth of the wildtype strain but not the mutant (Fig 2a, red hits).…”

Section: Exploiting Suppression Of Growth Inhibitory Activity To Tmentioning

confidence: 99%

Accelerating the discovery of antibacterial compounds using pathway-directed whole cell screening

Matano

Morris

Wood

et al. 2016

Bioorganic & Medicinal Chemistry

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Since the introduction of penicillin into the clinic in 1942, antibiotics have saved the lives of millions of people around the world. While penicillin and other traditional broad spectrum antibiotics were effective as monotherapies, the inexorable spread of antibiotic resistance has made alternative therapeutic approaches necessary. Compound combinations are increasingly seen as attractive options. Such combinations may include: lethal compounds; synthetically lethal compounds; or administering a lethal compound with a nonlethal compound that targets a virulence factor or a resistance factor. Regardless of the therapeutic strategy, high throughput screening is a key approach to discover potential leads. Unfortunately, the discovery of biologically active compounds that inhibit a desired pathway can be a very slow process, and an inordinate amount of time is often spent following up on compounds that do not have the desired biological activity. Here we describe a pathway-directed high throughput screening paradigm that combines the advantages of target-based and whole cell screens while minimizing the disadvantages. By exploiting this paradigm, it is possible to rapidly identify biologically active compounds that inhibit a pathway of interest. We describe some previous successful applications of this paradigm and report the discovery of a new class of D-alanylation inhibitors that may be useful as components of compound combinations to treat methicillin-resistant Staphylococcus aureus (MRSA).

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The Mechanism of Action of Lysobactin

Cited by 63 publications

References 36 publications

In vitro reconstitution demonstrates the cell wall ligase activity of LCP proteins

In vitro reconstitution demonstrates the cell wall ligase activity of LCP proteins

Identification of a Functionally Unique Family of Penicillin-Binding Proteins

Accelerating the discovery of antibacterial compounds using pathway-directed whole cell screening

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