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Identification of polyketide inhibitors targeting 3-dehydroquinate dehydratase in the shikimate pathway of Enterococcus faecalis.

Cheung VW, Xue B, Hernandez-Valladares M, Go MK, Tung A, Aguda AH, Robinson RC, Yew WS - PLoS ONE (2014)

Bottom Line: The Gram-positive commensal microbe, Enterococcus faecalis, is a major human pathogen associated with nosocomial infections and resistance to vancomycin, the "drug of last resort".The purification, crystallization and structural resolution of recombinant DHQase from E. faecalis (at 2.2 Å resolution) are also reported.This study provides a route in the development of polyketide-based antimicrobial inhibitors targeting the shikimate pathway of the human pathogen E. faecalis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

ABSTRACT
Due to the emergence of resistance toward current antibiotics, there is a pressing need to develop the next generation of antibiotics as therapeutics against infectious and opportunistic diseases of microbial origins. The shikimate pathway is exclusive to microbes, plants and fungi, and hence is an attractive and logical target for development of antimicrobial therapeutics. The Gram-positive commensal microbe, Enterococcus faecalis, is a major human pathogen associated with nosocomial infections and resistance to vancomycin, the "drug of last resort". Here, we report the identification of several polyketide-based inhibitors against the E. faecalis shikimate pathway enzyme, 3-dehydroquinate dehydratase (DHQase). In particular, marein, a flavonoid polyketide, both inhibited DHQase and retarded the growth of Enterococcus faecalis. The purification, crystallization and structural resolution of recombinant DHQase from E. faecalis (at 2.2 Å resolution) are also reported. This study provides a route in the development of polyketide-based antimicrobial inhibitors targeting the shikimate pathway of the human pathogen E. faecalis.

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efSHD-diaphorase coupled-enzyme assay.
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pone-0103598-g004: efSHD-diaphorase coupled-enzyme assay.

Mentions: Flavonoids that inhibited the efDHQase coupled-enzyme assay may inhibit efSHD instead of efDHQase. Thus, efSHD was assayed independently. efSHD was assayed using a continuous coupled-enzyme spectrophotometric assay (Figure 4) monitored at 25°C. The rate of 3-dehydroshikimate formation was coupled to the reduction of p-iodonitrotetrazolium (INT) (500 nm, ε = 17800 M−1 cm−1). The 200 µL assay contained 50 mM HEPES buffer, pH 7.5, 0.15 mM NADP+, 1.5 mM shikimate, 5 units of diaphorase (Sigma), 1 mM INT, and 10 nM efSHD. Flavonoids that did not affect the rate of dehydroshikimate formation were considered as potential efDHQase inhibitors.


Identification of polyketide inhibitors targeting 3-dehydroquinate dehydratase in the shikimate pathway of Enterococcus faecalis.

Cheung VW, Xue B, Hernandez-Valladares M, Go MK, Tung A, Aguda AH, Robinson RC, Yew WS - PLoS ONE (2014)

efSHD-diaphorase coupled-enzyme assay.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4114755&req=5

pone-0103598-g004: efSHD-diaphorase coupled-enzyme assay.
Mentions: Flavonoids that inhibited the efDHQase coupled-enzyme assay may inhibit efSHD instead of efDHQase. Thus, efSHD was assayed independently. efSHD was assayed using a continuous coupled-enzyme spectrophotometric assay (Figure 4) monitored at 25°C. The rate of 3-dehydroshikimate formation was coupled to the reduction of p-iodonitrotetrazolium (INT) (500 nm, ε = 17800 M−1 cm−1). The 200 µL assay contained 50 mM HEPES buffer, pH 7.5, 0.15 mM NADP+, 1.5 mM shikimate, 5 units of diaphorase (Sigma), 1 mM INT, and 10 nM efSHD. Flavonoids that did not affect the rate of dehydroshikimate formation were considered as potential efDHQase inhibitors.

Bottom Line: The Gram-positive commensal microbe, Enterococcus faecalis, is a major human pathogen associated with nosocomial infections and resistance to vancomycin, the "drug of last resort".The purification, crystallization and structural resolution of recombinant DHQase from E. faecalis (at 2.2 Å resolution) are also reported.This study provides a route in the development of polyketide-based antimicrobial inhibitors targeting the shikimate pathway of the human pathogen E. faecalis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

ABSTRACT
Due to the emergence of resistance toward current antibiotics, there is a pressing need to develop the next generation of antibiotics as therapeutics against infectious and opportunistic diseases of microbial origins. The shikimate pathway is exclusive to microbes, plants and fungi, and hence is an attractive and logical target for development of antimicrobial therapeutics. The Gram-positive commensal microbe, Enterococcus faecalis, is a major human pathogen associated with nosocomial infections and resistance to vancomycin, the "drug of last resort". Here, we report the identification of several polyketide-based inhibitors against the E. faecalis shikimate pathway enzyme, 3-dehydroquinate dehydratase (DHQase). In particular, marein, a flavonoid polyketide, both inhibited DHQase and retarded the growth of Enterococcus faecalis. The purification, crystallization and structural resolution of recombinant DHQase from E. faecalis (at 2.2 Å resolution) are also reported. This study provides a route in the development of polyketide-based antimicrobial inhibitors targeting the shikimate pathway of the human pathogen E. faecalis.

Show MeSH
Related in: MedlinePlus