Identification of an alternative nucleoside triphosphate: 5'-deoxyadenosylcobinamide phosphate nucleotidyltransferase in Methanobacterium thermoautotrophicum Delta H

Computer analysis of the archaeal genome databases failed to identify ortho logues of all of the bacterial cobamide biosynthetic enzymes. Of particular interest was the lack of an orthologue of the bifunctional nucleoside trip hosphate (NTP):5'-deoxyadenosylcobinamide kinase/GTP:adenosylcobinamide-pho sphate guanylyltransferase enzyme (CobU in Salmonella enterica). This paper reports the identification of an archaeal gene encoding a new nucleotidylt ransferase, which is proposed to be the nonorthologous replacement of the S . enterica cobU gene. The gene encoding this nucleotidyltransferase was ide ntified 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 fro m Methanobacterium thermoautotrophicum Delta H (MTH1152; referred to as cob Y) was amplified and cloned, and the CobY protein was expressed and purifie d from Escherichia coli as a hexahistidine tagged fusion protein. This enzy me had GTP:adenosylcobinamide-phosphate guanylyltransferase activity but di d not have the NTP:AdoCbi kinase activity associated with the CobU enzyme o f S. enterica. NTP:adenosylcobinamide kinase activity,vas not detected in M . thermoautotrophicum Delta H cell extract, suggesting that this organism m ay not have this activity. The cobY gene complemented a cobU mutant of S. e nterica 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 conditi ons where de novo synthesis of this coenzyme was blocked, and growth of the cell depended on the assimilation of exogenous cobinamide. These data stro ngly support the proposal that the relevant cobinamide intermediates during de novo adenosylcobalamin biosynthesis are adenosylcobinamide-phosphate an d adenosylcobinamide-GDP, not adenosylcobinamide. Therefore, NTP:adenosylco binamide kinase activity is not required for de novo cobamide biosynthesis.