| Autor: |
Mattes TA; Department of Microbiology, University of Georgia, Athens, Georgia 30602, United States., Malalasekara L; Department of Microbiology, University of Georgia, Athens, Georgia 30602, United States., Escalante-Semerena JC; Department of Microbiology, University of Georgia, Athens, Georgia 30602, United States. |
| Jazyk: |
angličtina |
| Zdroj: |
Biochemistry [Biochemistry] 2021 Jun 29; Vol. 60 (25), pp. 2011-2021. Date of Electronic Publication: 2021 Jun 09. |
| DOI: |
10.1021/acs.biochem.1c00119 |
| Abstrakt: |
We report the initial characterization of the α-ribazole (α-R) kinase enzyme of Geobacillus kaustophilus ( Gk CblS), 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 K m 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 Gk CblS activated α-R and other α-ribosides, including α-adenosine (α-Ado). Gk CblS 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 Gk CblS 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 Gk CblS has specificity for α-N-glycosidic bonds, but not for the base in α-ribosides. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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