Targeting an Aromatic Hotspot in <i>Plasmodium falciparum</i> 1‐Deoxy‐<scp>d</scp>‐xylulose‐5‐phosphate Reductoisomerase with β‐Arylpropyl Analogues of Fosmidomycin

Sooriyaarachchi, Sanjeewani; Chofor, René; Risseeuw, Martijn; Bergfors, Terese; Pouyez, Jenny; Dowd, Cynthia S.; Maes, Louis; Jones, T. Alwyn; Calenbergh, Serge Van; Mowbray, Sherry L.

doi:10.1002/cmdc.201600249

Cited by 20 publications

(16 citation statements)

References 46 publications

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“…Similar degradation of a related buffer component 3-(N-morpholino)propanesulfonic acid (MOPS) and binding of morpholine to the protein was observed by Sooriyaarachchi et al . during crystallization of Plasmodium falciparum 1-deoxy-d-xylulose-5-phosphate Reductoisomerase [24] (PDB-ID 5JO0). Interestingly, the protein also crystallized using another precipitant solution for which we obtained crystals in two crystal forms.…”

Section: Resultsmentioning

confidence: 99%

Structural and mechanistic insight into spectral tuning in flavin-binding fluorescent proteins

Röllen

Granzin

Remeeva

et al. 2021

Preprint

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Determining the molecular origin of spectral tuning in photoactive biological systems is instrumental for understanding their function. Spectral-tuning efforts for flavin-binding fluorescent proteins (FbFPs), an emerging class of fluorescent reporters, are limited by their dependency on protein-bound flavins, whose structure and hence electronic properties, cannot be altered by mutation. To address those shortcomings, we here present the photophysical, computational and structural characterization of structurally uncharacterized blue-shifted FbFPs, carrying a previously described lysine substitution within their flavin-binding pocket. X-ray structures reveal displacement of the lysine away from the chromophore and opening up of the structure as cause for the blue shift. Site-saturation mutagenesis and high-throughput screening, yielded a red-shifted variant, in which the lysine side chain of the blue-shifted variant is stabilized in close distance to the flavin by a secondary mutation, mechanistically accounting for the red shift. Thus, a single secondary mutation in a blue-shifted variant is sufficient to generate a red-shifted FbFP. Using spectroscopy, X-ray crystallography and quantum mechanics molecular mechanics calculations, we provide a firm structural and functional understanding of spectral tuning in FbFPs. We also show that the identified blue- and red-shifted variants allow for two-color microscopy based on spectral separation. In summary, the generated blue- and red-shifted variants represent promising new tools that should find application in life sciences.

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Section: Resultsmentioning

confidence: 99%

Structural and mechanistic insight into spectral tuning in flavin-binding fluorescent proteins

Röllen

Granzin

Remeeva

et al. 2021

Preprint

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“…A DALI search identified multiple DXR from Escherichia coli, Plasmodium falciparum, M. tuberculosis , and other microbes (Z-scores: 49-51; r.m.s.d. ∼1.6 Å 2 for 370-400 C α -atoms; 39-40% amino acid sequence identity)(31–36). The monomer consists of three regions (Fig 3A): an N-terminal α/β-domain with a central 7-stranded β-sheet (β1-β7) and 7 α-helices that serves as the nucleotide binding site; a middle region of the protein that includes a second β-sheet (β8-β11) and 4 α-helices (α8 and α12-α14); and a C-terminal α-helical domain (α9-α11 and α15-α18) that locks FSM into the active site(37).…”

Section: Resultsmentioning

confidence: 99%

“…Movement of this flexible loop is a key feature for FSM inhibition of DXR from a variety of microorganisms(38). The residues that interact with FSM in the S. schleiferi DXR are conserved in the crystal structures of DXR from E. coli, P. falciparum , and M. tuberculosis with some variation in the sequence of the α10-α11 loop, although the tryptophan that contacts FSM is conserved in all these enzymes(34,36,37).…”

Section: Resultsmentioning

confidence: 99%

Potent, specific MEPicides for treatment of zoonotic staphylococci

Edwards¹,

Heueck²,

Lee

et al. 2019

Preprint

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24Coagulase-positive staphylococci, which frequently colonize the mucosal surfaces of 25 animals, also cause a spectrum of opportunistic infections including skin and soft tissue 26 infections, urinary tract infections, pneumonia, and bacteremia. However, recent 27 45 robustly inhibit DXR in zoonotic staphylococci, and further, DXR represents a 46 promising, druggable target for future development. 3 47 48 Author Summary 49 The proliferation of microbial pathogens resistant to the current pool of antibiotics is a 50 major threat to public health. Drug resistance is pervasive in staphylococci, including 51 several species that can cause serious zoonotic infections in humans. Thus, new 52 antimicrobial agents are urgently need to combat these life-threatening, resistant 53 infections. Here we establish the MEP pathway as a promising new target against 54 zoonotic staphylococci. We determine that fosmidomycin (FSM) selectively targets the 55 isoprenoid biosynthesis pathway in zoonotic staphylococci, and use forward genetics to 56 identify the transporter that facilitates phosphonate antibiotic uptake. Employing this 57 knowledge, we synthesized a series of potent antibacterial prodrugs that circumvent 58 the transporter. Together, these novel prodrug inhibitors represent promising leads for 59 further drug development against zoonotic staphylococci. 60 61 65 zoonotic infections in humans that are clinically indistinguishable from infections with S. 66 aureus including pneumonia, skin and soft tissue infections, hardware infections, and 67 bacteremia(1-5). Newer clinical microbiological techniques, such as mass 68 spectrometry, now readily distinguish S. aureus from zoonotic coagulase-positive

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“…Further, we confirm that DXR inhibition by FSM is competitive with respect to the DOXP substrate, with a K i [DOXP] of 0.29 ± 0.022 μM ( Fig 3B). https://doi.org/10.1371/journal.ppat.1007806.g001 [31][32][33][34][35][36]. The monomer consists of three regions ( Fig 4A): an N-terminal α/β-domain with a central 7-stranded β-sheet (β1-β7) and 7 α-helices that serves as the nucleotide binding site; a middle region of the protein that includes a second β-sheet (β8-β11) and 4 α-helices (α8 and α12-α14); and a C-terminal α-helical domain (α9-α11 and α15-α18) that locks FSM into the active site [37].…”

Section: Fosmidomycin Is a Competitive Inhibitor Of S Schleiferi Dxrmentioning

confidence: 99%

“…E. coli, P. falciparum, and M. tuberculosis, with some variation in the sequence of the α10-α11 loop, although the tryptophan that contacts FSM is conserved in all these enzymes [34,36,37].…”

Section: Plos Pathogensmentioning

confidence: 99%

Potent, specific MEPicides for treatment of zoonotic staphylococci

et al. 2020

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Coagulase-positive staphylococci, which frequently colonize the mucosal surfaces of animals, also cause a spectrum of opportunistic infections including skin and soft tissue infections, urinary tract infections, pneumonia, and bacteremia. However, recent advances in bacterial identification have revealed that these common veterinary pathogens are in fact zoonoses that cause serious infections in human patients. The global spread of multidrugresistant zoonotic staphylococci, in particular the emergence of methicillin-resistant organisms, is now a serious threat to both animal and human welfare. Accordingly, new therapeutic targets that can be exploited to combat staphylococcal infections are urgently needed. Enzymes of the methylerythritol phosphate pathway (MEP) of isoprenoid biosynthesis represent potential targets for treating zoonotic staphylococci. Here we demonstrate that fosmidomycin (FSM) inhibits the first step of the isoprenoid biosynthetic pathway catalyzed by deoxyxylulose phosphate reductoisomerase (DXR) in staphylococci. In addition, we have both enzymatically and structurally determined the mechanism by which FSM elicits its effect. Using a forward genetic screen, the glycerol-3-phosphate transporter GlpT that facilitates FSM uptake was identified in two zoonotic staphylococci, Staphylococcus schleiferi and Staphylococcus pseudintermedius. A series of lipophilic ester prodrugs (termed PLOS PATHOGENS

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Targeting an Aromatic Hotspot in Plasmodium falciparum 1‐Deoxy‐d‐xylulose‐5‐phosphate Reductoisomerase with β‐Arylpropyl Analogues of Fosmidomycin

Cited by 20 publications

References 46 publications

Structural and mechanistic insight into spectral tuning in flavin-binding fluorescent proteins

Structural and mechanistic insight into spectral tuning in flavin-binding fluorescent proteins

Potent, specific MEPicides for treatment of zoonotic staphylococci

Potent, specific MEPicides for treatment of zoonotic staphylococci

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