Functional Studies of α-Riboside Activation by the α-Ribazole Kinase (CblS) from Geobacillus kaustophilus

Biochemistry. 2021 Jun 29;60(25):2011-2021. doi: 10.1021/acs.biochem.1c00119. Epub 2021 Jun 9.


We report the initial characterization of the α-ribazole (α-R) kinase enzyme of Geobacillus kaustophilus (GkCblS), which converts α-R to α-R-phosphate (α-RP) during the synthesis of cobamides. We implemented a continuous spectrophotometric assay to obtain kinetic parameters for several potential substrates and to study the specificity of the enzyme for α-N-linked ribosides. The apparent Km values for α-R and ATP were 358 and 297 μM, respectively. We also report methods for synthesizing and quantifying non-commercially available α-ribosides and β-ribazole (β-R). Purified GkCblS activated α-R and other α-ribosides, including α-adenosine (α-Ado). GkCblS did not phosphorylate β-N-linked glycosides like β-adenosine or β-R. Expression of G. kaustophilus cblS+ in a Salmonella enterica subsp. enterica sv Typhimurium LT2 (S. enterica) strain lacking the nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyl transferase (CobT) enzyme resulted in the activation of various benzimidazole α-ribosides, and the synthesis of benzimidazolyl cobamides to levels that supported robust growth. Notably, α-Ado did not support growth under similar conditions, in spite of the fact that GkCblS phosphorylated α-Ado in vitro. When α-Ado was provided at a very high concentration, growth was observed. This result suggested that in S. enterica α-Ado transport may be inefficient. We conclude that GkCblS has specificity for α-N-glycosidic bonds, but not for the base in α-ribosides.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / isolation & purification
  • Enzyme Assays
  • Geobacillus / enzymology*
  • Kinetics
  • Phosphorylation
  • Phosphotransferases (Alcohol Group Acceptor) / chemistry*
  • Phosphotransferases (Alcohol Group Acceptor) / isolation & purification
  • Purine-Nucleoside Phosphorylase / chemistry
  • Ribonucleosides / chemical synthesis
  • Ribonucleosides / chemistry*
  • Salmonella / enzymology
  • Substrate Specificity


  • Bacterial Proteins
  • Ribonucleosides
  • alpha-ribazole
  • Purine-Nucleoside Phosphorylase
  • Phosphotransferases (Alcohol Group Acceptor)

Supplementary concepts

  • Geobacillus kaustophilus
  • Salmonella enterica subsp. enterica