title: Identification of an alternative nucleoside triphosphate: 5'-deoxyadenosylcobinamide phosphate nucleotidyltransferase in Methanobacterium thermoautotrophicum delta H.
abstract: Computer analysis of the archaeal genome databases failed to identify orthologues of all of the bacterial cobamide biosynthetic enzymes. Of particular interest was the lack of an orthologue of the bifunctional nucleoside triphosphate (NTP):5'-deoxyadenosylcobinamide kinase/GTP:adenosylcobinamide-phosphate guanylyltransferase enzyme (CobU in Salmonella enterica). This paper reports the identification of an archaeal gene encoding a new nucleotidyltransferase, which is proposed to be the nonorthologous replacement of the S. enterica cobU gene. The gene encoding this nucleotidyltransferase was identified using comparative genome analysis of the sequenced archaeal genomes. Orthologues of the gene encoding this activity are limited at present to members of the domain Archaea. The corresponding ORF open reading frame from Methanobacterium thermoautotrophicum Delta H (MTH1152; referred to as cobY) was amplified and cloned, and the CobY protein was expressed and purified from Escherichia coli as a hexahistidine-tagged fusion protein. This enzyme had GTP:adenosylcobinamide-phosphate guanylyltransferase activity but did not have the NTP:AdoCbi kinase activity associated with the CobU enzyme of S. enterica. NTP:adenosylcobinamide kinase activity was not detected in M. thermoautotrophicum Delta H cell extract, suggesting that this organism may not have this activity. The cobY gene complemented a cobU mutant of S. enterica grown under anaerobic conditions where growth of the cell depended on de novo adenosylcobalamin biosynthesis. cobY, however, failed to restore adenosylcobalamin biosynthesis in cobU mutants grown under aerobic conditions where de novo synthesis of this coenzyme was blocked, and growth of the cell depended on the assimilation of exogenous cobinamide. These data strongly support the proposal that the relevant cobinamide intermediates during de novo adenosylcobalamin biosynthesis are adenosylcobinamide-phosphate and adenosylcobinamide-GDP, not adenosylcobinamide. Therefore, NTP:adenosylcobinamide kinase activity is not required for de novo cobamide biosynthesis.
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706-1567, USA.
authors.forename: M G
authors.initials: MG
authors.lastname: Thomas
authors.forename: J C
authors.initials: JC
authors.lastname: Escalante-Semerena
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D002384:Catalysis; D003038:Cobamides / Q000096:biosynthesis / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D017014:Methanobacterium / Q000201:enzymology*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D016366:Open Reading Frames; D010430:Pentosyltransferases / Q000235:genetics / Q000378:metabolism*; D019779:Salmonella enterica / Q000201:enzymology
chemical_list: D019843:Archaeal Proteins; D003038:Cobamides; D009097:Multienzyme Complexes; C412035:guanosine 5'-(5'-deoxyadenosylcobinamide pyrophosphate); D006153:Guanosine Diphosphate; C079634:adenosylcobinamide methyl phosphate; D010430:Pentosyltransferases; C028015:nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase; D009713:Nucleotidyltransferases; C412034:cobY protein, Methanobacterium thermoautotrophicum; C000913:cobamamide