Inactivation of Lactobacillus leichmannii ribonucleotide reductase by F2CTP: covalent modification
Ribonucleotide reductase (RNR) from Lactobacillus leichmannii, a 76 kDa monomer using adenosylcobalamin (AdoCbl) as a cofactor, catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is rapidly (<30 s) inactivated by 1 equiv of 2′,2′-difluoro-2′-deoxycytidine 5′-triphosphate...
| Main Authors: | , |
|---|---|
| Other Authors: | |
| Format: | Article |
| Language: | en_US |
| Published: |
American Chemical Society (ACS)
2012
|
| Online Access: | http://hdl.handle.net/1721.1/73105 https://orcid.org/0000-0001-8076-4489 |
| _version_ | 1826205853443686400 |
|---|---|
| author | Lohman, Gregory J. S. Stubbe, JoAnne |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Lohman, Gregory J. S. Stubbe, JoAnne |
| author_sort | Lohman, Gregory J. S. |
| collection | MIT |
| description | Ribonucleotide reductase (RNR) from Lactobacillus leichmannii, a 76 kDa monomer using adenosylcobalamin (AdoCbl) as a cofactor, catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is rapidly (<30 s) inactivated by 1 equiv of 2′,2′-difluoro-2′-deoxycytidine 5′-triphosphate (F[subscript 2]CTP). [1′-[superscript 3]H]- and [5-[superscript 3]H]F[subscript 2]CTP were synthesized and used independently to inactivate RNR. Sephadex G-50 chromatography of the inactivation mixture revealed that 0.47 equiv of a sugar was covalently bound to RNR and that 0.71 equiv of cytosine was released. Alternatively, analysis of the inactivated RNR by SDS−PAGE without boiling resulted in 33% of RNR migrating as a 110 kDa protein. Inactivation of RNR with a mixture of [1′-[superscript 3]H]F[subscript 2]CTP and [1′-[superscript 2]H]F[subscript 2]CTP followed by reduction with NaBH[subscript 4], alkylation with iodoacetamide, trypsin digestion, and HPLC separation of the resulting peptides allowed isolation and identification by MALDI-TOF mass spectrometry (MS) of a 3H/2H-labeled peptide containing C[subscript 731] and C[subscript 736] from the C-terminus of RNR accounting for 10% of the labeled protein. The MS analysis also revealed that the two cysteines were cross-linked to a furanone species derived from the sugar of F[subscript 2]CTP. Incubation of [1′-[superscript 3]H]F[subscript 2]CTP with C119S-RNR resulted in 0.3 equiv of sugar being covalently bound to the protein, and incubation with NaBH[subscript 4] subsequent to inactivation resulted in trapping of 2′-fluoro-2′-deoxycytidine. These studies and the ones in the preceding paper (DOI: 10.1021/bi9021318) allow proposal of a mechanism of inactivation of RNR by F[subscript 2]CTP involving multiple reaction pathways. The proposed mechanisms share many common features with F[subscript 2]CDP inactivation of the class I RNRs. |
| first_indexed | 2024-09-23T13:20:08Z |
| format | Article |
| id | mit-1721.1/73105 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T13:20:08Z |
| publishDate | 2012 |
| publisher | American Chemical Society (ACS) |
| record_format | dspace |
| spelling | mit-1721.1/731052022-10-01T14:35:20Z Inactivation of Lactobacillus leichmannii ribonucleotide reductase by F2CTP: covalent modification Lohman, Gregory J. S. Stubbe, JoAnne Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Chemistry Lohman, Gregory J. S. Stubbe, JoAnne Ribonucleotide reductase (RNR) from Lactobacillus leichmannii, a 76 kDa monomer using adenosylcobalamin (AdoCbl) as a cofactor, catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is rapidly (<30 s) inactivated by 1 equiv of 2′,2′-difluoro-2′-deoxycytidine 5′-triphosphate (F[subscript 2]CTP). [1′-[superscript 3]H]- and [5-[superscript 3]H]F[subscript 2]CTP were synthesized and used independently to inactivate RNR. Sephadex G-50 chromatography of the inactivation mixture revealed that 0.47 equiv of a sugar was covalently bound to RNR and that 0.71 equiv of cytosine was released. Alternatively, analysis of the inactivated RNR by SDS−PAGE without boiling resulted in 33% of RNR migrating as a 110 kDa protein. Inactivation of RNR with a mixture of [1′-[superscript 3]H]F[subscript 2]CTP and [1′-[superscript 2]H]F[subscript 2]CTP followed by reduction with NaBH[subscript 4], alkylation with iodoacetamide, trypsin digestion, and HPLC separation of the resulting peptides allowed isolation and identification by MALDI-TOF mass spectrometry (MS) of a 3H/2H-labeled peptide containing C[subscript 731] and C[subscript 736] from the C-terminus of RNR accounting for 10% of the labeled protein. The MS analysis also revealed that the two cysteines were cross-linked to a furanone species derived from the sugar of F[subscript 2]CTP. Incubation of [1′-[superscript 3]H]F[subscript 2]CTP with C119S-RNR resulted in 0.3 equiv of sugar being covalently bound to the protein, and incubation with NaBH[subscript 4] subsequent to inactivation resulted in trapping of 2′-fluoro-2′-deoxycytidine. These studies and the ones in the preceding paper (DOI: 10.1021/bi9021318) allow proposal of a mechanism of inactivation of RNR by F[subscript 2]CTP involving multiple reaction pathways. The proposed mechanisms share many common features with F[subscript 2]CDP inactivation of the class I RNRs. National Institutes of Health (U.S.) (Grant number GM-29595) 2012-09-21T17:54:17Z 2012-09-21T17:54:17Z 2010-02 2010-01 Article http://purl.org/eprint/type/JournalArticle 0006-2960 1520-4995 http://hdl.handle.net/1721.1/73105 Lohman, Gregory J. S., and JoAnne Stubbe. “Inactivation of Lactobacillus Leichmannii Ribonucleotide Reductase by 2′,2′-Difluoro-2′-deoxycytidine 5′-Triphosphate: Covalent Modification.” Biochemistry 49.7 (2010): 1404–1417. https://orcid.org/0000-0001-8076-4489 en_US http://dx.doi.org/10.1021/bi902132u Biochemistry Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf American Chemical Society (ACS) PMC |
| spellingShingle | Lohman, Gregory J. S. Stubbe, JoAnne Inactivation of Lactobacillus leichmannii ribonucleotide reductase by F2CTP: covalent modification |
| title | Inactivation of Lactobacillus leichmannii ribonucleotide reductase by F2CTP: covalent modification |
| title_full | Inactivation of Lactobacillus leichmannii ribonucleotide reductase by F2CTP: covalent modification |
| title_fullStr | Inactivation of Lactobacillus leichmannii ribonucleotide reductase by F2CTP: covalent modification |
| title_full_unstemmed | Inactivation of Lactobacillus leichmannii ribonucleotide reductase by F2CTP: covalent modification |
| title_short | Inactivation of Lactobacillus leichmannii ribonucleotide reductase by F2CTP: covalent modification |
| title_sort | inactivation of lactobacillus leichmannii ribonucleotide reductase by f2ctp covalent modification |
| url | http://hdl.handle.net/1721.1/73105 https://orcid.org/0000-0001-8076-4489 |
| work_keys_str_mv | AT lohmangregoryjs inactivationoflactobacillusleichmanniiribonucleotidereductasebyf2ctpcovalentmodification AT stubbejoanne inactivationoflactobacillusleichmanniiribonucleotidereductasebyf2ctpcovalentmodification |