title: The last step in coenzyme B(12) synthesis is localized to the cell membrane in bacteria and archaea.
abstract: In Salmonella enterica, the last step of the synthesis of adenosylcobamide is catalysed by the cobalamin synthase enzyme encoded by the cobS gene of this bacterium. Overexpression of the S. enterica cobS gene in Escherichia coli elicited the accumulation of the phage shock protein PspA, a protein whose expression has been linked to membrane stress. Resolution of inner and outer membranes of S. enterica by isopycnic density ultracentrifugation showed CobS activity associated with the inner membrane, a result that was confirmed using antibodies against CobS. Computer analysis of the predicted amino acid sequence of CobS suggested it was an integral membrane protein. Results of experiments performed with strains carrying plasmids encoding CobS-alkaline phosphatase or CobS-beta-galactosidase protein fusions were consistent with the membrane localization of the CobS protein. Modifications to the predicted model were made based on data obtained from experiments using protein fusions. The function encoded by the cobS orthologue in the methanogenic archaeon Methanobacterium thermoautotrophicum strain deltaH compensated for the lack of CobS during cobalamin synthesis in cobS strains of S. enterica. Cobalamin synthase activity was also detected in a membrane preparation of M. thermoautotrophicum. It was concluded that the assembly of the nucleotide loop of adenosylcobamides in archaea and bacteria is a membrane-associated process. Possible reasons for the association of adenosylcobamide biosynthetic enzymes with the cell membrane are discussed.
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, 1710 University Avenue, Madison, WI 53726-4087, USA.
authors.forename: Lori A
authors.initials: LA
authors.lastname: Maggio-Hall
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, 1710 University Avenue, Madison, WI 53726-4087, USA.
authors.forename: Kathy R
authors.initials: KR
authors.lastname: Claas
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, 1710 University Avenue, Madison, WI 53726-4087, USA.
authors.forename: Jorge C
authors.initials: JC
authors.lastname: Escalante-Semerena
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology*; D003038:Cobamides / Q000096:biosynthesis* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D017014:Methanobacterium / Q000201:enzymology*; D019884:Nitrogenous Group Transferases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019779:Salmonella enterica / Q000201:enzymology*; D014461:Ultracentrifugation
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D003038:Cobamides; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D019884:Nitrogenous Group Transferases; C000913:cobamamide