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.

FIGURE 1

UV-vis spectra of AdoCbl analog(s) bound to RTPR (solid line) and in solution (dashed line) subsequent to Sephadex G50 chromatography of RTPR inactivated by F₂CTP in the presence of 1 eq. of AdoCbl. (A) after 2 min. (B) after 1 h.

FIGURE 2

Reverse-phase HPLC of small molecules from RTPR inactivated by F₂CTP, treated with NaBH₄, and dephosphorylated with alkaline phosphatase. A(260) (solid line), HPLC gradient (˙˙˙˙˙˙): Buffer A, 10 mM NH₄OAc, pH 6.8; Buffer B: methanol. The double headed arrow indicates the region pooled in each step. (A) Initial purification: the material eluting from 17–22 min was collected. (B) Elution of final purified product after two further repurifications under the same conditions. (C) Reverse-phase HPLC of small molecules from RTPR inactivated with F₂CTP, treated with NaBD₄, and dephosphorylated with alkaline phosphatase. This is the second step in the three step purification.

FIGURE 3

Comparison of ¹H-NMR (500 MHz, D₂O) spectra of new products isolated from the NaBH₄ (NaB₂H₄) quench of RTPR inactivated with F₂CTP. (A) Product from NaBH₄ quench; (B) product from NaB₂H₄ quench. (A′ and B′ are expansions of A and B in the region from 3.5 to 4.5 ppm). The nucleoside proton resonances are labeled. Several impurity peaks can be seen; these are marked with X. Further, the H5–H5′ region in B appears to overlap with an impurity peak.

FIGURE 4

High Frequency (130 GHz) Davies ENDOR of F₂CTP reacted with RTPR for 30 s. (A) Deuterium ENDOR of [1′-²H]-F₂CTP reaction. Temperature 7 K; 8000 averages per point; pulse widths 80 ns, 40 ns, 80 ns; RF pulse width 20 µs, tau 200 ns; Rep rate 100 Hz (B) Proton ENDOR. Temperature 7 K; pulse widths 70 ns, 35ns 70ns ; RF pulse width 6.8 µs; tau 200 ns; Rep rate 100 Hz Upper trace: RTPR reacted with [1′-¹H]-F₂CTP, 800 averages per point. The peaks at 169 and 226 MHz are indicated. Lower Trace: RTPR reacted with [1′-²H]-F₂CTP, 4500 averages per point

SCHEME 1

Proposed model for the mechanism of inactivation by RTPR by F₂CTP by the non-alkylative and alkylative pathways.

SCHEME 2

Proposed mechanism for deuterium incorporation into the nucleotide trapped by NaB₂H₄ subsequent to the inactivation of RTPR by F₂CTP.