title: Structural and functional investigation into acetyl-coenzyme A synthase and methyltransferase from human pathogen Clostridium difficile.
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.
authors.affiliation: Department of Chemistry & Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Xiaofei
authors.initials: X
authors.lastname: Zhu
authors.forename: Tiejun
authors.initials: T
authors.lastname: Li
authors.forename: Xiang
authors.initials: X
authors.lastname: Gu
authors.forename: Sixue
authors.initials: S
authors.lastname: Zhang
authors.forename: Yi
authors.initials: Y
authors.lastname: Liu
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.forename: Xiangshi
authors.initials: X
authors.lastname: Tan
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D055162:Biocatalysis / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D016360:Clostridioides difficile / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005453:Fluorescence; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007700:Kinetics; D008780:Methyltransferases / Q000737:chemistry* / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D005810:Multigene Family; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D008780:Methyltransferases; D003066:Coenzyme A Ligases