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

We report the initial characterization of the aribazole (alpha-R) kinase enzyme of Geobacillus kaustophilus (GkCblS), which converts alpha-R to alpha-R-phosphate (alpha-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 alpha-N-linked ribosides. The apparent Km values for alpha-R and ATP were 358 and 297 mu M, respectively. We also report methods for synthesizing and quantifying non-commercially available alpha-ribosides and beta-ribazole (beta-R). Purified GkCblS activated alpha-R and other alpha-ribosides, including alpha-adenosine (alpha-Ado). GkCblS did not phosphorylate beta-N-linked glycosides like beta-adenosine or beta-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 alpha-ribosides, and the synthesis of benzimidazolyl cobamides to levels that supported robust growth. Notably, alpha-Ado did not support growth under similar conditions, in spite of the fact that GkCblS phosphorylated alpha-Ado in vitro. When alpha-Ado was provided at a very high concentration, growth was observed. This result suggested that in S. enterica alpha-Ado transport may be inefficient. We conclude that GkCblS has specificity for alpha-N-glycosidic bonds, but not for the base in alpha-ribosides.

Automated summary

The study reports an enzyme from Geobacillus kaustophilus that converts alpha-R to alpha-R-phosphate and exhibits specificity for certain alpha-N-linked ribosides. The enzyme does not phosphorylate beta-N-linked glycosides and alpha-adenosine is unable to support growth under certain conditions, despite being phosphorylated by the enzyme in vitro.