title: Efficient expression and purification of methyltransferase in acetyl-coenzyme a synthesis pathway of the human pathogen Clostridium difficile.
abstract: The Wood-Ljungdahl pathway is responsible for acetyl-CoA biosynthesis and used as a major mean of generating energy for growth in some anaerobic microbes. Series of genes, from the anaerobic human pathogen Clostridium difficile, have been identified that show striking similarity to the genes involved in this pathway including methyltetrahydrofolate- and corrinoid-dependent methyltransferase. This 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 C. difficile for the first time with two expression vectors MBPHT-mCherry2 and pETDuet-1, respectively. Using the latter vector, more than 50mg MeTr was produced per liter Luria-Bertani broth media. The recombinant methyltransferase was well characterized by SDS-PAGE, gel filtration chromatography, enzyme assay and far-UV circular dichroism (CD). Furthermore, a highly effective approach was established for determining the methyl transfer activity of the methyltetrahydrofolate- and cobalamin-dependent methyltransferase using exogenous cobalamin as a substrate by stopped-flow method. These results will provide a solid basis for further study of the methyltransferase and the Wood-Ljungdahl pathway.
authors.affiliation: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Xiaofei
authors.initials: X
authors.lastname: Zhu
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: Zhong-Xian
authors.initials: ZX
authors.lastname: Huang
authors.forename: Xiangshi
authors.initials: X
authors.lastname: Tan
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D016360:Clostridioides difficile / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006879:Hydroxocobalamin; D007700:Kinetics; D008780:Methyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D000105:Acetyl Coenzyme A; D008780:Methyltransferases; D006879:Hydroxocobalamin