The cbiS gene of the archaeon Methanopyrus kandleri AV19 encodes a bifunctional enzyme with adenosylcobinamide amidohydrolase and alpha-ribazole-phosphate phosphatase activities.
Here we report the initial biochemical characterization of the bifunctional alpha-ribazole-P (alpha-RP) phosphatase, adenosylcobinamide (AdoCbi) amidohydrolase CbiS enzyme from the hyperthermophilic methanogenic archaeon Methanopyrus kandleri AV19. The cbiS gene encodes a 39-kDa protein with two distinct segments, one of which is homologous to the AdoCbi amidohydrolase (CbiZ, EC 3.5.1.90) enzyme and the other of which is homologous to the recently discovered archaeal alpha-RP phosphatase (CobZ, EC 3.1.3.73) enzyme. CbiS function restored AdoCbi salvaging and alpha-RP phosphatase activity in strains of the bacterium Salmonella enterica where either step was blocked. The two halves of the cbiS genes retained their function in vivo when they were cloned separately. The CbiS enzyme was overproduced in Escherichia coli and was isolated to >95% homogeneity. High-performance liquid chromatography, UV-visible spectroscopy, and mass spectroscopy established alpha-ribazole and cobyric acid as the products of the phosphatase and amidohydrolase reactions, respectively. Reasons why the CbiZ and CobZ enzymes are fused in some archaea are discussed.
Woodson
Jesse D
JD
144A Enzyme Institute, 1710 University Avenue, Madison, WI 53726-4087. escalante@bact.wisc.edu.
Escalante-Semerena
Jorge C
JC
D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism*; D001105:Archaea / Q000201:enzymology* / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism; D050939:Gene Fusion; D019766:Genes, Archaeal / Q000235:genetics; D005816:Genetic Complementation Test; D008970:Molecular Weight; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D012263:Ribonucleosides / Q000032:analysis / Q000378:metabolism; D019779:Salmonella enterica / Q000235:genetics / Q000378:metabolism; D013057:Spectrum Analysis; D014805:Vitamin B 12 / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism
D011994:Recombinant Proteins; D012263:Ribonucleosides; C015491:alpha-ribazole; C012851:cobyric acid; D010744:Phosphoric Monoester Hydrolases; D000581:Amidohydrolases; D014805:Vitamin B 12