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Issue 5, 2013
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Structural and functional investigation into acetyl-coenzyme A synthase and methyltransferase from human pathogen Clostridium difficile

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Abstract

Methyltransferase (MeTrCd) and acetyl-coenzyme A synthase (ACSCd) are two key enzymes in the acetyl-coenzyme A synthesis pathway of the human pathogen Clostridium difficile. The pathway is absent in humans and is essential for the survival of the pathogen. MeTrCd and ACSCd were cloned, expressed in E. coli, and characterized for the first time. Structural and functional investigations of the two enzymes were performed using homology structure modeling, fluorescence spectroscopy, and steady state/pre-steady state kinetics. The conformational change and methyl transfer activity of MeTrCd were shown to be pH dependent. The kinetic studies of MeTrCd at the optimal pH 5.1 yield the parameters kcat (2.63 s−1), Km (17.8 μM) and kcat/Km (0.15 μM−1 s−1). The active site metal cluster (A-cluster) of ACSCd, [Fe4S4][NipNid], was characterized using metal analysis, structural modeling, and UV/Vis spectra of the characteristic features of [Fe4S4] cubane. Nip, as a labile metal, can be removed by treatment with chelators, resulting in the loss of ACS activity. Three bidentate chelators (1,10-phenanthroline, 8-hydroxyquinoline, and 2,2-dipyridyl) exhibited excellent inhibition effects on ACSCd methyl group transfer and acetyl-coenzyme A synthesis activity. These inhibitory effects were further examined using antibacterial activity assays against Clostridium difficile. These results provide a new strategy to find new potential antibiotics for the treatment of CDI.

Graphical abstract: Structural and functional investigation into acetyl-coenzyme A synthase and methyltransferase from human pathogen Clostridium difficile

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Publication details

The article was received on 20 Dec 2012, accepted on 02 Apr 2013 and first published on 03 Apr 2013


Article type: Paper
DOI: 10.1039/C3MT20257G
Metallomics, 2013,5, 551-558

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    Structural and functional investigation into acetyl-coenzyme A synthase and methyltransferase from human pathogen Clostridium difficile

    X. Zhu, T. Li, X. Gu, S. Zhang, Y. Liu, Y. Wang and X. Tan, Metallomics, 2013, 5, 551
    DOI: 10.1039/C3MT20257G

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