Inactivation of lactobacillus leichmannii ribonucleotide reductase by 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate

Adenosylcobalamin destruction and formation of a nucleotide-based radical

Gregory J S Lohman, Gary J. Gerfen, Joanne Stubbe

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Ribonucleotide reductase (RNR, 76 kDa) from Lactobacillus leichmannii is a class II RNR that requires adenosylcobalamin (AdoCbl) as a cofactor. It catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is 100% inactivated by 1 equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F2CTP) in < 2 min. Sephadex G-50 chromatography of the inactivation reaction mixture for 2 min revealed that 0.47 equiv of a sugar moiety is covalently bound to RNR and 0.25 equiv of a cobalt(III) corrin is tightly associated, likely through a covalent interaction with C419 (Co-S) in the active site of RNR [Lohman, G. J. S., and Stubbe, J. (2010) Biochemistry 49, DOI: 10.1021/bi902132u]. After 1 h, a similar experiment revealed 0.45 equiv of the Co-S adduct associated with the protein. Thus, at least two pathways are associated with RNR inactivation: one associated with alkylation on by the sugar Of F2CTP and the second with AdoCbl destruction. To determine the fate of [1'-3H]F2CTP in the latter pathway, the reaction mixture at 2 min was reduced with NaB2H 4 (NaB2H4) and the protein separated from the small molecules using a centrifugation device. The small molecules were dephosphorylated and analyzed by HPLC to reveal 0.25 equiv of a stereoisomer of cytidine, characterized by mass spectrometry and NMR spectroscopy, indicating the trapped nucleotide had lost both of its fluorides and gained an oxygen. High-field ENDOR studies with [I'- 2H]F2CTP from the reaction quenched at 30 s revealed a radical that is nucleotide-based. The relationship between this radical and the trapped cytidine analogue provides insight into the nonalkylative pathway for RNR inactivation relative to the alkylative pathway.

Original languageEnglish (US)
Pages (from-to)1396-1403
Number of pages8
JournalBiochemistry
Volume49
Issue number7
DOIs
StatePublished - Feb 23 2010

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Lactobacillus leichmannii
Ribonucleotide Reductases
Cytidine
Sugars
Mass Spectrometry
Nucleotides
Biochemistry
Molecules
Stereoisomerism
Centrifugation
Alkylation
Electron Spin Resonance Spectroscopy
Chromatography
Cobalt
Fluorides
Nucleosides
Nuclear magnetic resonance spectroscopy
Mass spectrometry
Catalytic Domain
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Inactivation of lactobacillus leichmannii ribonucleotide reductase by 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate: Adenosylcobalamin destruction and formation of a nucleotide-based radical",
abstract = "Ribonucleotide reductase (RNR, 76 kDa) from Lactobacillus leichmannii is a class II RNR that requires adenosylcobalamin (AdoCbl) as a cofactor. It catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is 100{\%} inactivated by 1 equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F2CTP) in < 2 min. Sephadex G-50 chromatography of the inactivation reaction mixture for 2 min revealed that 0.47 equiv of a sugar moiety is covalently bound to RNR and 0.25 equiv of a cobalt(III) corrin is tightly associated, likely through a covalent interaction with C419 (Co-S) in the active site of RNR [Lohman, G. J. S., and Stubbe, J. (2010) Biochemistry 49, DOI: 10.1021/bi902132u]. After 1 h, a similar experiment revealed 0.45 equiv of the Co-S adduct associated with the protein. Thus, at least two pathways are associated with RNR inactivation: one associated with alkylation on by the sugar Of F2CTP and the second with AdoCbl destruction. To determine the fate of [1'-3H]F2CTP in the latter pathway, the reaction mixture at 2 min was reduced with NaB2H 4 (NaB2H4) and the protein separated from the small molecules using a centrifugation device. The small molecules were dephosphorylated and analyzed by HPLC to reveal 0.25 equiv of a stereoisomer of cytidine, characterized by mass spectrometry and NMR spectroscopy, indicating the trapped nucleotide had lost both of its fluorides and gained an oxygen. High-field ENDOR studies with [I'- 2H]F2CTP from the reaction quenched at 30 s revealed a radical that is nucleotide-based. The relationship between this radical and the trapped cytidine analogue provides insight into the nonalkylative pathway for RNR inactivation relative to the alkylative pathway.",
author = "Lohman, {Gregory J S} and Gerfen, {Gary J.} and Joanne Stubbe",
year = "2010",
month = "2",
day = "23",
doi = "10.1021/bi9021318",
language = "English (US)",
volume = "49",
pages = "1396--1403",
journal = "Biochemistry",
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T1 - Inactivation of lactobacillus leichmannii ribonucleotide reductase by 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate

T2 - Adenosylcobalamin destruction and formation of a nucleotide-based radical

AU - Lohman, Gregory J S

AU - Gerfen, Gary J.

AU - Stubbe, Joanne

PY - 2010/2/23

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N2 - Ribonucleotide reductase (RNR, 76 kDa) from Lactobacillus leichmannii is a class II RNR that requires adenosylcobalamin (AdoCbl) as a cofactor. It catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is 100% inactivated by 1 equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F2CTP) in < 2 min. Sephadex G-50 chromatography of the inactivation reaction mixture for 2 min revealed that 0.47 equiv of a sugar moiety is covalently bound to RNR and 0.25 equiv of a cobalt(III) corrin is tightly associated, likely through a covalent interaction with C419 (Co-S) in the active site of RNR [Lohman, G. J. S., and Stubbe, J. (2010) Biochemistry 49, DOI: 10.1021/bi902132u]. After 1 h, a similar experiment revealed 0.45 equiv of the Co-S adduct associated with the protein. Thus, at least two pathways are associated with RNR inactivation: one associated with alkylation on by the sugar Of F2CTP and the second with AdoCbl destruction. To determine the fate of [1'-3H]F2CTP in the latter pathway, the reaction mixture at 2 min was reduced with NaB2H 4 (NaB2H4) and the protein separated from the small molecules using a centrifugation device. The small molecules were dephosphorylated and analyzed by HPLC to reveal 0.25 equiv of a stereoisomer of cytidine, characterized by mass spectrometry and NMR spectroscopy, indicating the trapped nucleotide had lost both of its fluorides and gained an oxygen. High-field ENDOR studies with [I'- 2H]F2CTP from the reaction quenched at 30 s revealed a radical that is nucleotide-based. The relationship between this radical and the trapped cytidine analogue provides insight into the nonalkylative pathway for RNR inactivation relative to the alkylative pathway.

AB - Ribonucleotide reductase (RNR, 76 kDa) from Lactobacillus leichmannii is a class II RNR that requires adenosylcobalamin (AdoCbl) as a cofactor. It catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is 100% inactivated by 1 equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F2CTP) in < 2 min. Sephadex G-50 chromatography of the inactivation reaction mixture for 2 min revealed that 0.47 equiv of a sugar moiety is covalently bound to RNR and 0.25 equiv of a cobalt(III) corrin is tightly associated, likely through a covalent interaction with C419 (Co-S) in the active site of RNR [Lohman, G. J. S., and Stubbe, J. (2010) Biochemistry 49, DOI: 10.1021/bi902132u]. After 1 h, a similar experiment revealed 0.45 equiv of the Co-S adduct associated with the protein. Thus, at least two pathways are associated with RNR inactivation: one associated with alkylation on by the sugar Of F2CTP and the second with AdoCbl destruction. To determine the fate of [1'-3H]F2CTP in the latter pathway, the reaction mixture at 2 min was reduced with NaB2H 4 (NaB2H4) and the protein separated from the small molecules using a centrifugation device. The small molecules were dephosphorylated and analyzed by HPLC to reveal 0.25 equiv of a stereoisomer of cytidine, characterized by mass spectrometry and NMR spectroscopy, indicating the trapped nucleotide had lost both of its fluorides and gained an oxygen. High-field ENDOR studies with [I'- 2H]F2CTP from the reaction quenched at 30 s revealed a radical that is nucleotide-based. The relationship between this radical and the trapped cytidine analogue provides insight into the nonalkylative pathway for RNR inactivation relative to the alkylative pathway.

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