Functional Studies of α-Riboside Activation by the α-Ribazole Kinase (CblS) from Geobacillus kaustophilus.
Biochemistry
; 60(25): 2011-2021, 2021 06 29.
Article
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| MEDLINE
| ID: mdl-34105957
ABSTRACT
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 ß-ribazole (ß-R). Purified GkCblS activated α-R and other α-ribosides, including α-adenosine (α-Ado). GkCblS did not phosphorylate ß-N-linked glycosides like ß-adenosine or ß-R. Expression of G. kaustophilus cblS+ in a Salmonella enterica subsp. enterica sv Typhimurium LT2 (S. enterica) strain lacking the nicotinate mononucleotide5,6-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.
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Banco de datos:
MEDLINE
Asunto principal:
Ribonucleósidos
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Proteínas Bacterianas
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Fosfotransferasas (Aceptor de Grupo Alcohol)
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Geobacillus
Idioma:
En
Año:
2021
Tipo del documento:
Article