Inactivation of Lactobacillus leichmannii ribonucleotide reductase by F2CTP: 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[subscript 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[subscript 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[subscript 2]CTP and the second with AdoCbl destruction. To determine the fate of [1′-[superscript 3]H]F2CTP in the latter pathway, the reaction mixture at 2 min was reduced with NaBH[subscript 4] (NaB[superscript 2]H[subscript 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′-[superscript 2]H]F[subscript 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.