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Joanne Stubbe - One of the best experts on this subject based on the ideXlab platform.
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inactivation of Lactobacillus leichmannii ribonucleotide reductase by 2 2 difluoro 2 deoxycytidine 5 triphosphate covalent modification
Biochemistry, 2010Co-Authors: Gregory J S Lohman, Joanne StubbeAbstract: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 (F2CTP). [1′-3H]- and [5-3H]F2CTP 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′-3H]F2CTP and [1′-2H]F2CTP followed by reduction with NaBH4, 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 C731 and C736 from the ...
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inactivation of Lactobacillus leichmannii ribonucleotide reductase by 2 2 difluoro 2 deoxycytidine 5 triphosphate adenosylcobalamin destruction and formation of a nucleotide based radical
Biochemistry, 2010Co-Authors: Gregory J S Lohman, Gary J Gerfen, Joanne StubbeAbstract: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 by the sugar of F2CTP and the second with AdoCbl destruction. To determine the fate of [1′-3H]F2CTP in the latter pathway, the...
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The crystal structure of class II ribonucleotide reductase reveals how an allosterically regulated monomer mimics a dimer
Nature Structural Biology, 2002Co-Authors: Michael D. Sintchak, Joanne Stubbe, Gitrada Arjara, Brenda A. Kellogg, Catherine L. DrennanAbstract:Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides, an essential step in DNA biosynthesis and repair. Here we present the crystal structure of class II (coenzyme B_12-dependent) ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii in the apo enzyme form and in complex with the B_12 analog adeninylpentylcobalamin at 1.75 and 2.0 Å resolution, respectively. This monomeric, allosterically regulated class II RNR retains all the key structural features associated with the catalytic and regulatory machinery of oligomeric RNRs. Surprisingly, the dimer interface responsible for effector binding in class I RNR is preserved through a single 130-residue insertion in the class II structure. Thus, L. leichmannii RNR is a paradigm for the simplest structural entity capable of ribonucleotide reduction, a reaction linking the RNA and DNA worlds.
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binding of cob ii alamin to the adenosylcobalamin dependent ribonucleotide reductase from Lactobacillus leichmannii identification of dimethylbenzimidazole as the axial ligand
Journal of Biological Chemistry, 1999Co-Authors: Christopher C Lawrence, Gary J Gerfen, Vicente Samano, Rainer Nitsche, Morris J Robins, Janos Retey, Joanne StubbeAbstract:Abstract The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of nucleoside 5′-triphosphates to 2′-deoxynucleoside 5′-triphosphates and uses coenzyme B12, adenosylcobalamin (AdoCbl), as a cofactor. Use of a mechanism-based inhibitor, 2′-deoxy-2′-methylenecytidine 5′-triphosphate, and isotopically labeled RTPR and AdoCbl in conjunction with EPR spectroscopy has allowed identification of the lower axial ligand of cob(II)alamin when bound to RTPR. In common with the AdoCbl-dependent enzymes catalyzing irreversible heteroatom migrations and in contrast to the enzymes catalyzing reversible carbon skeleton rearrangements, the dimethylbenzimidazole moiety of the cofactor is not displaced by a protein histidine upon binding to RTPR.
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gemcitabine 5 triphosphate is a stoichiometric mechanism based inhibitor of Lactobacillus leichmannii ribonucleoside triphosphate reductase evidence for thiyl radical mediated nucleotide radical formation
Biochemistry, 1998Co-Authors: Domingos J Silva, Joanne Stubbe, Vicente Samano, Morris J RobinsAbstract:Ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii utilizes adenosylcobalamin and catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates. One equivalent of 2‘,2‘-difluoro-2‘-deoxycytidine 5‘-triphosphate, F2dCTP, rapidly inactivates RTPR. Analysis of the reaction products reveals that inactivation is accompanied by release of two fluoride ions and 0.84 equiv of 5‘-deoxyadenosine and attachment of 1 equiv of corrin covalently to an active-site cysteine residue of RTPR. No cytosine release was detected. Proteolysis of corrin-labeled RTPR with endoproteinase Glu-C and peptide mapping at pH 5.8 revealed that C419 was predominantly modified. The kinetics of the inactivation have been examined by stopped-flow (SF) UV−vis spectroscopy and rapid freeze quench (RFQ) electron paramagnetic resonance (EPR) spectroscopy. Monitoring ΔA525 nm shows that cob(II)alamin is formed with an apparent kobs of 50 s-1, only 2.5-fold slower than a similar experiment carried o...
Gwyn P. Jones - One of the best experts on this subject based on the ideXlab platform.
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Comparison of a competitive binding assay with Lactobacillus leichmannii A.T.C.C. 7830 assay for the determination of vitamin B12 in foods
Food Chemistry, 1993Co-Authors: Kharidah Muhammad, David R. Briggs, Gwyn P. JonesAbstract:Abstract The results obtained by a competitive binding method for the determination of vitamin B 12 in food were compared with those obtained by a widely used microbiological assay with Lactobacillus leichmannii A.T.C.C. 7830. Both assays were performed on the same sample extract. The extraction was carried out at pH 4.5, 121°C for 10 min with 0.1 m sodium acetate-acetic acid buffer in the presence of potassium cyanide. A high correlation ( r 2 = 0.841) between the results from the two methods was found. In the case of pork and yoghurt, the large differences observed could be due to the presence of substances in their extracts which interfere with the assays. The competitive binding assay can therefore be applied to the determination of vitamin B 12 in some foods.
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the appropriateness of using cyanocobalamin as calibration standards in Lactobacillus leichmannii a t c c 7830 assay of vitamin b12
Food Chemistry, 1993Co-Authors: Kharidah Muhammad, David R. Briggs, Gwyn P. JonesAbstract:Abstract The appropriateness of using solutions of cyanocobalamin as the calibration standards in Lactobacillus leichmannii A.T.C.C. 7830 assay of total vitamin B 12 in foods, plasma and serum was examined. This is because the forms of the vitamins that may be present after the addition of sodium cyanide, potassium cyanide, sodium metabisulphite or sodium nitrite in the sample extraction procedure are hydroxocobalamin, sulphitocobalamin, cyanocobalamin, adenosylcobalamin, methylcobalamin, dicyanocobalamin and nitritocobalamin, depending on the form of endogenous vitamin B 12 . Therefore, if Lactobacillus leichmannii A.T.C.C. 7830 has a higher or lower growth response to cyanocobalamin than to the other potential forms of cobalamin, the amount of vitamin B 12 determined by comparisons with calibration standards prepared from cyanocobalamin will be an under- or over-estimation. The results of this study showed that the growth response of Lactobacillus leichmannii A.T.C.C. 7830 to cyanocobalamin was similar to its growth responses to hydroxocobalamin, sulphitocobalamin, dicyanocobalamin and nitritocobalamin but lower than that to adenosylcobalamin and higher than that to methylcobalamin. Thus, total vitamin B 12 cannot be measured accurately using Lactobacillus leichmannii A.T.C.C. 7830 assay which employs cyanocobalamin as its calibration standards if adenosylcobalamin or methylcobalamin is present.
Felix Zelder - One of the best experts on this subject based on the ideXlab platform.
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inorganic cyanide as protecting group in the stereospecific reconstitution of vitamin b12 from an artificial green secocorrinoid
Organic Letters, 2016Co-Authors: Lucas Prieto, Markus Neuburger, Bernhard Spingler, Felix ZelderAbstract:The synthesis of vitamin B12 in four steps from an artificial green secocorrinoid is presented. The stereospecific reconstitution of the B-ring of the cobalamin involves a quantitative and rapid ligand-centered radical ring closure reaction leading first to a new B12 derivative with antivitamin activity that is subsequently converted to the natural product. Chemoselectivity in the one-electron reduction of the macrocycle was achieved by introducing inorganic cyanide as an axially coordinating protecting group of the otherwise reduction-sensitive CoIII-ion. The integrity of structure and function of the reconstituted natural product was unequivocally proven by single crystal structural analysis and a microbiological assay using Lactobacillus leichmannii.
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Inorganic Cyanide as Protecting Group in the Stereospecific Reconstitution of Vitamin B12 from an Artificial Green Secocorrinoid
2016Co-Authors: Lucas Prieto, Markus Neuburger, Bernhard Spingler, Felix ZelderAbstract:The synthesis of vitamin B12 in four steps from an artificial green secocorrinoid is presented. The stereospecific reconstitution of the B-ring of the cobalamin involves a quantitative and rapid ligand-centered radical ring closure reaction leading first to a new B12 derivative with antivitamin activity that is subsequently converted to the natural product. Chemoselectivity in the one-electron reduction of the macrocycle was achieved by introducing inorganic cyanide as an axially coordinating protecting group of the otherwise reduction-sensitive CoIII-ion. The integrity of structure and function of the reconstituted natural product was unequivocally proven by single crystal structural analysis and a microbiological assay using Lactobacillus leichmannii
Kharidah Muhammad - One of the best experts on this subject based on the ideXlab platform.
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Comparison of a competitive binding assay with Lactobacillus leichmannii A.T.C.C. 7830 assay for the determination of vitamin B12 in foods
Food Chemistry, 1993Co-Authors: Kharidah Muhammad, David R. Briggs, Gwyn P. JonesAbstract:Abstract The results obtained by a competitive binding method for the determination of vitamin B 12 in food were compared with those obtained by a widely used microbiological assay with Lactobacillus leichmannii A.T.C.C. 7830. Both assays were performed on the same sample extract. The extraction was carried out at pH 4.5, 121°C for 10 min with 0.1 m sodium acetate-acetic acid buffer in the presence of potassium cyanide. A high correlation ( r 2 = 0.841) between the results from the two methods was found. In the case of pork and yoghurt, the large differences observed could be due to the presence of substances in their extracts which interfere with the assays. The competitive binding assay can therefore be applied to the determination of vitamin B 12 in some foods.
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the appropriateness of using cyanocobalamin as calibration standards in Lactobacillus leichmannii a t c c 7830 assay of vitamin b12
Food Chemistry, 1993Co-Authors: Kharidah Muhammad, David R. Briggs, Gwyn P. JonesAbstract:Abstract The appropriateness of using solutions of cyanocobalamin as the calibration standards in Lactobacillus leichmannii A.T.C.C. 7830 assay of total vitamin B 12 in foods, plasma and serum was examined. This is because the forms of the vitamins that may be present after the addition of sodium cyanide, potassium cyanide, sodium metabisulphite or sodium nitrite in the sample extraction procedure are hydroxocobalamin, sulphitocobalamin, cyanocobalamin, adenosylcobalamin, methylcobalamin, dicyanocobalamin and nitritocobalamin, depending on the form of endogenous vitamin B 12 . Therefore, if Lactobacillus leichmannii A.T.C.C. 7830 has a higher or lower growth response to cyanocobalamin than to the other potential forms of cobalamin, the amount of vitamin B 12 determined by comparisons with calibration standards prepared from cyanocobalamin will be an under- or over-estimation. The results of this study showed that the growth response of Lactobacillus leichmannii A.T.C.C. 7830 to cyanocobalamin was similar to its growth responses to hydroxocobalamin, sulphitocobalamin, dicyanocobalamin and nitritocobalamin but lower than that to adenosylcobalamin and higher than that to methylcobalamin. Thus, total vitamin B 12 cannot be measured accurately using Lactobacillus leichmannii A.T.C.C. 7830 assay which employs cyanocobalamin as its calibration standards if adenosylcobalamin or methylcobalamin is present.
Gary J Gerfen - One of the best experts on this subject based on the ideXlab platform.
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investigating the intermediates in the reaction of ribonucleoside triphosphate reductase from Lactobacillus leichmannii an application of hf epr rfq technology
Journal of Magnetic Resonance, 2011Co-Authors: Julia Manzerova, Vladimir Krymov, Gary J GerfenAbstract:Abstract In this investigation high-frequency electron paramagnetic resonance spectroscopy (HFEPR) in conjunction with innovative rapid freeze-quench (RFQ) technology is employed to study the exchange-coupled thiyl radical–cob(II)alamin system in ribonucleotide reductase from a prokaryote Lactobacillus leichmannii. The size of the exchange coupling (Jex) and the values of the thiyl radical g tensor are refined, while confirming the previously determined (Gerfen et al. (1996) [20] ) distance between the paramagnets. Conclusions relevant to ribonucleotide reductase catalysis and the architecture of the active site are presented. A key part of this work has been the development of a unique RFQ apparatus for the preparation of millisecond quench time RFQ samples which can be packed into small (0.5 mm ID) sample tubes used for CW and pulsed HFEPR – lack of this ability has heretofore precluded such studies. The technology is compatible with a broad range of spectroscopic techniques and can be readily adopted by other laboratories.
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inactivation of Lactobacillus leichmannii ribonucleotide reductase by 2 2 difluoro 2 deoxycytidine 5 triphosphate adenosylcobalamin destruction and formation of a nucleotide based radical
Biochemistry, 2010Co-Authors: Gregory J S Lohman, Gary J Gerfen, Joanne StubbeAbstract: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 by the sugar of F2CTP and the second with AdoCbl destruction. To determine the fate of [1′-3H]F2CTP in the latter pathway, the...
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binding of cob ii alamin to the adenosylcobalamin dependent ribonucleotide reductase from Lactobacillus leichmannii identification of dimethylbenzimidazole as the axial ligand
Journal of Biological Chemistry, 1999Co-Authors: Christopher C Lawrence, Gary J Gerfen, Vicente Samano, Rainer Nitsche, Morris J Robins, Janos Retey, Joanne StubbeAbstract:Abstract The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of nucleoside 5′-triphosphates to 2′-deoxynucleoside 5′-triphosphates and uses coenzyme B12, adenosylcobalamin (AdoCbl), as a cofactor. Use of a mechanism-based inhibitor, 2′-deoxy-2′-methylenecytidine 5′-triphosphate, and isotopically labeled RTPR and AdoCbl in conjunction with EPR spectroscopy has allowed identification of the lower axial ligand of cob(II)alamin when bound to RTPR. In common with the AdoCbl-dependent enzymes catalyzing irreversible heteroatom migrations and in contrast to the enzymes catalyzing reversible carbon skeleton rearrangements, the dimethylbenzimidazole moiety of the cofactor is not displaced by a protein histidine upon binding to RTPR.
