Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis

Hevener, Kirk E.; Mehboob, Shahila; Boci, Teuta; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E.

doi:10.1016/j.pep.2012.07.003

Cited by 7 publications

(10 citation statements)

References 29 publications

(34 reference statements)

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“…It is more difficult to predict the behavior of the Bacteroidia class of organisms because FabI and FabK are both found in organisms belonging to this class. Based on sequenced genomes of family members (NCBI Microbial Genomes Resources), members of the Prevotellaceae family encode only a FabI, members of the Porphyromonadaceae family encode only a FabK (37), and the Bacteroidaceae and Rikenellaceae families encode both FabI and FabK. The observation that Debio 1452 does not reduce the abundance of the Rikenellaceae family, whose members are predicted to encode both FabI and FabK, indicates that the FabK enoyl-ACP reductase compensates for a Debio 1452-inactivated FabI.…”

Section: Figmentioning

confidence: 99%

A Pathogen-Selective Antibiotic Minimizes Disturbance to the Microbiome

Yao

Carter

Vuagniaux

et al. 2016

Antimicrob Agents Chemother

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c Broad-spectrum antibiotic therapy decimates the gut microbiome, resulting in a variety of negative health consequences. Debio 1452 is a staphylococcus-selective enoyl-acyl carrier protein reductase (FabI) inhibitor under clinical development and was used to determine whether treatment with pathogen-selective antibiotics would minimize disturbance to the microbiome. The effect of oral Debio 1452 on the microbiota of mice was compared to the effects of four commonly used broad-spectrum oral antibiotics. During the 10 days of oral Debio 1452 treatment, there was minimal disturbance to the gut bacterial abundance and composition, with only the unclassified S24-7 taxon reduced at days 6 and 10. In comparison, broad-spectrum oral antibiotics caused ϳ100-to 4,000-fold decreases in gut bacterial abundance and severely altered the microbial composition. The gut bacterial abundance and composition of Debio 1452-treated mice were indistinguishable from those of untreated mice 2 days after the antibiotic treatment was stopped. In contrast, the bacterial abundance in broad-spectrum-antibiotic-treated mice took up to 7 days to recover, and the gut composition of the broad-spectrum-antibiotic-treated mice remained different from that of the control group 20 days after the cessation of antibiotic treatment. These results illustrate that a pathogen-selective approach to antibiotic development will minimize disturbance to the gut microbiome.

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Section: Figmentioning

confidence: 99%

A Pathogen-Selective Antibiotic Minimizes Disturbance to the Microbiome

Yao

Carter

Vuagniaux

et al. 2016

Antimicrob Agents Chemother

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“…The FabK enzyme from Streptococcus pneumoniae ( Sp FabK) has been previously characterized (Marrakchi et al , 2003) and structures, both apo and with a known inhibitor bound, have been determined for this organism, leading to several key observations (Saito et al , 2008, Saito et al , 2006). Sp FabK is a flavoenzyme, dependent upon an FMN prosthetic group and NADH cofactor, and uses a ‘Ping-Pong’, bi-bi enzyme mechanism to reduce the enoyl substrate (Hevener et al , 2012, Saito et al , 2008). Sp FabK is structurally distinct from FabI (as well as FabV and FabL) and possesses an overall triose-phosphate isomerase (TIM) barrel structural fold, whereas FabI, FabV and FabL fall within the short-chain dehydrogenase/reductase (SDR) superfamily of reductases and possess a classical Rossmann fold for binding to their NAD(P)H cofactor (White et al , 2005, Oppermann et al , 2003).…”

Section: Introductionmentioning

confidence: 99%

Structural characterization ofPorphyromonas gingivalisenoyl-ACP reductase II (FabK)

Hevener¹,

Santarsiero²,

Lee³

et al. 2018

Acta Cryst Sect F

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Enoyl-acyl carrier protein (ACP) reductase II (FabK) is a critical rate-limiting enzyme in the bacterial type II fatty-acid synthesis (FAS II) pathway. FAS II pathway enzymes are markedly disparate from their mammalian analogs in the FAS I pathway in both structure and mechanism. Enzymes involved in bacterial fatty-acid synthesis represent viable drug targets for Gram-negative pathogens, and historical precedent exists for targeting them in the treatment of diseases of the oral cavity. The Gram-negative organism Porphyromonas gingivalis represents a key causative agent of the costly and highly prevalent disease known as chronic periodontitis, and exclusively expresses FabK as its enoyl reductase enzyme in the FAS-II pathway. Together, these characteristics distinguish P. gingivalis FabK (PgFabK) as an attractive and novel narrow-spectrum antibacterial target candidate. PgFabK is a flavoenzyme that is dependent on FMN and NADPH as cofactors for the enzymatic reaction, which reduces the enoyl substrate via a ping-pong mechanism. Here, the structure of the PgFabK enzyme as determined using X-ray crystallography is reported to 1.9 Å resolution with endogenous FMN fully resolved and the NADPH cofactor partially resolved. PgFabK possesses a TIM-barrel motif, and all flexible loops are visible. The determined structure has allowed insight into the structural basis for the NADPH dependence observed in PgFabK and the role of a monovalent cation that has been observed in previous studies to be stringently required for FabK activity. The PgFabK structure and the insights gleaned from its analysis will facilitate structure-based drug-discovery efforts towards the prevention and treatment of P. gingivalis infection.

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“…It also suggests that while FabK is a functional dimer, its active sites function independently. 33,37 Inhibition of FASII reduces spore production.…”

Section: Difficile Fabk Is Druggable Targetmentioning

confidence: 99%

“…Susceptibility to test compounds was determined by microbroth dilution in 96-well microtiter plates under anaerobic growth condition as described. 37 For S. pyogenes and S. aureus aerobic growth conditions were maintained. The MIC was determined as the lowest compound concentration inhibiting visible growth at 24 h.…”

Section: Susceptibility Tests and Effects On Growthmentioning

confidence: 99%

“…A composite homology model of the CdFabK enzyme was created using the published structures of the S. pneumoniae (pdb ID 2z6j) and Porphyromonas gingivalis (pdb ID 4IQL) FabK enzymes as templates. 33,37 P. gingivalis loop positions were used for missing loop regions in the S. pneumoniae template. The Prime software package available in the Schrödinger molecular modeling suite (Schrödinger Release 2018-1: Prime, Schrödinger, LLC, New York, NY,USA) was used to build and refine the model.…”

Section: Computational Modelling Of Cdfabkzmentioning

confidence: 99%

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The Fatty Acid Synthesis Protein Enoyl-ACP Reductase II (FabK) is a Target for Narrow-Spectrum Antibacterials for Clostridium difficile Infection

Marreddy

Sapkota

et al. 2018

ACS Infect. Dis.

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Clostridium difficile infection (CDI) is an antibiotic-induced microbiota shift disease of the large bowel. While there is a need for narrow-spectrum CDI antibiotics, it is unclear which cellular proteins are appropriate drug targets to specifically inhibit C. difficile. We evaluated the enoyl-acyl carrier protein reductase II (FabK) that catalyzes the final step of bacterial fatty acid biosynthesis. Bioinformatics showed C. difficile uses FabK as its sole enoyl-ACP reductase, unlike several major microbiota species. The essentiality of fabK for C. difficile growth was confirmed by failure to delete this gene using Clostron mutagenesis and by growth inhibition upon gene silencing with CRISPR-interference antisense to fabK transcription or by blocking protein translation. Inhibition of C. difficile's FASII pathway could not be circumvented by supply of exogenous fatty acids, either during fabK's gene silencing or upon inhibition of the enzyme with a phenylimidazolederived inhibitor 1. The inability for fatty acids to bypass FASII inhibition is likely due to the function of transcriptional repressor FapR. Inhibition of FabK also inhibited spore formation, reflecting the enzyme's role in de novo fatty acid biosynthesis for the formation of spore membrane lipids. Compound 1 did not inhibit growth of key microbiota species. These findings suggest that C. difficile FabK is a druggable target for discovering narrow-spectrum anti-C. difficile drugs to treat CDI but avoid collateral damage to the gut microbiota.

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Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis

Cited by 7 publications

References 29 publications

A Pathogen-Selective Antibiotic Minimizes Disturbance to the Microbiome

A Pathogen-Selective Antibiotic Minimizes Disturbance to the Microbiome

Structural characterization ofPorphyromonas gingivalisenoyl-ACP reductase II (FabK)

The Fatty Acid Synthesis Protein Enoyl-ACP Reductase II (FabK) is a Target for Narrow-Spectrum Antibacterials for Clostridium difficile Infection

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