| Clostridium difficile is a ubiquitous anaerobic and spore-forming pathogen. It can produce toxins A and B, and then causes acute illnesses called Clostridium difficile infection (CDI) in humans, ranging from severe diarrhea, antibiotic-associated colitis, pseudomembranous colitis, toxic megacolon, intestinal perforations, and even death. It was found that the genomic DNA of the pathogen contains several genes encoding proteins involved in acetyl-coenzyme A synthesis of Wood-Ljungdahl pathway. The pathway for catabolism and anabolism of the pathogen involves a series of critical enzymes such as formate dehydrogenase, corrinoid-dependent methyltransferase, corrinoid iron-sulfur protein, and acetyl-CoA synthase. Each of the four metalloenzymes plays a critical role in the Wood-Ljungdahl pathway. Thus, these metalloenzymes could be a selective and promising target for the development of new antibiotics agents. Compounds targeting this enzyme should avoid multiple resistance with currently used antibiotics agents. It could be a new strategy to find special antibiotics for the treatment of CDI.In this study, we investigated some key metalloenzymes including methyltransferase (MeTrCd,268 aa), Corrinoid iron-sulfur proteins (CoFeSPCd, two subunits included:delta,314 aa and gamma,455 aa), acetyl-coenzyme A synthase (ACSCd,708 aa) and carbon monoxide dehydrogenases (CODHCd,639 aa).Corrinoid-dependent methyltransferase plays a central role in this pathway that transfers the methyl group from methyltetrahydrofolate to a cob(I)amide center in the corrinoid iron-sulfur protein. In this study, we developed two efficient expression and purification methods for methyltransferase from Clostridium difficile for the first time with two expression vectors MBPHT-mCherry2 and pETDuet-1, respectively. Using the latter vector, more than 50 mg MeTr was produced per liter TB medium. The recombinant methyltransferase was well characterized by SDS-PAGE, gel filtration chromatograpgy, enzyme assay and far-UV circular dichroism (CD). Furthermore, steady-state kinetics were studied using exogenous cobalamin and CoFeSP from Moorella thermoacetica (CoFeSPMt) as a substrate by stopped-flow method. MeTrCd showed pH dependent conformational change and methyl transfer activity. The kinetic studies of MeTrCd were fitted to the Michaelis-Menten equation, yielding values of kcat(77.6 s-1) and kcat/Km (1.33?M-1s-1) for hydroxycobalamin, and kcat(2.63 s-1) and kcat/Km(0.15?M-1s-1) for CoFeSPMt.CoFeSP acts as a transformer in the Wood-Ljungdahl pathway, which belongs to B12-dependent enzymes. In this study, the putative gamma and delta gene of CoFeSPcd from clostridium difficile was firstly expressed in E. coli. High yield of recombinant gammaCd and deltaCd was achieved in this expression system, which was purified efficiently by affinity chromatography. Furthermore, we got the holo-protein using the reconstitution of Fe4S4 and cobalamin. Then, steady-state kinetic was studied using CoFeSPCd as a methyl accepter by stopped-flow method, yielding values of kcat(43.7 s-1) and kcat/Km (0.51?M-s-1). The results of kinetic parameters, using hydroxycobalamin, CoFeSPMt and CoFeSPCd as a substrate respectively, were suggested that the interaction between CoFeSP and MeTr was a key step in the methyl transfer reaction.Acetyl-CoA synthases catalyses the condensation of three substrates:CoA, CO, and a methyl group from the methylated corrinoid iron-sulfur protein (CH3-CoFeSP), to produce acetyl-CoA. In this study, the target gene was subcloned into the vector pET30a (Novagen) to generate a C-terminal His-tagged protein. Based this, we established a recombinant expression system for ACSCd in E. coli that enabled a one-step purification of active enzyme under anaerobic conditions. The A-cluster, [Fe4S4][NipNid], existed in ACSCd was confirmed by structural modeling, metal analysis, and UV-Vis spectra of the characteristic feature of [Fe4S4] cubane. The Nip, as a labile metal, can be removed by treatment with chelators but this treatment results in the loss of ACS activity. Three bidentate chelators (10-phenanthroline, 8-hydroxyquinoline, and 2,2-dipyridyl) showed inhibition effects on ACSCd methyl group transfer and acetyl-coenzyme A synthesis activity. These inhibitory results were confirmed by further antibacterial activity assay against Clostridium difficile, though 8-hydroxyquinoline has a weak effect. These results played a solid base for the future research of high throughput screening to find valuable inhibitors, and it might be a new strategy to find new antibiotics for the treatment of CDI.At last, we tried to establish a recombinant expression and purifaction system for CODH. CODH catalyzes the reversible oxidation and reduction between carbon monoxide and carbon dioxide. CODHs from Carboxydothermus hydrogenoformans, Rhodospirillum rubrum and Moorella thermoacetica have been well studied. X-ray crystal structures of all three enzymes (CODHRr, CODHCh and CODHMt) were determined between 2001 and 2007. However, the reaction mechanism is probably the most intriguing. We constructed several plasmids and tried to establish a highly effective expression and purification system for CODHmt and CODHCd.Luckily, we got the MBP-fusion protein. All these studies will provide useful information for future research.In summary, we described for the first time several means of expressing at high level and purifying methyltetrahydrofolate- and corrinoid-dependent methyl-transferase, corrinoid iron-sulfur protein and acetyl-coenzyme A synthase from human pathogen C. difficile. Their structures and functions were investigated by spectroscopy, enzyme kinetics, and homology structure modeling. Inhibitors based on the enzyme ACSCd assay showed effective antibacterial activity for the pathogen C. difficile. In particular, this study provides a solid basis for the studies of potential drug target like ACSCd, which may apply to drug discovery for the treatment of CDI. |
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