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 (F(2)CTP) 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 C(419) (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 F(2)CTP and the second with AdoCbl destruction. To determine the fate of [1'-(3)H]F(2)CTP in the latter pathway, the reaction mixture at 2 min was reduced with NaBH(4) (NaB(2)H(4)) 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 [1'-(2)H]F(2)CTP 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.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Gregory J S
authors.initials: GJ
authors.lastname: Lohman
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Gerfen
authors.forename: Joanne
authors.initials: J
authors.lastname: Stubbe
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D003038:Cobamides / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000633:toxicity; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000633:toxicity; D005609:Free Radicals / Q000378:metabolism; D052199:Lactobacillus leichmannii / Q000201:enzymology*; D008956:Models, Chemical; D009705:Nucleosides / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D012266:Ribose / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D003038:Cobamides; D004791:Enzyme Inhibitors; D005609:Free Radicals; D009705:Nucleosides; D009711:Nucleotides; C054403:2',2'-difluorodeoxycytidine 5'-triphosphate; D003570:Cytidine Triphosphate; D012266:Ribose; D012264:Ribonucleotide Reductases; C000913:cobamamide