Human cytidine deaminase: a biochemical characterization of its naturally occurring variants

Abstract

Human cytidine deaminase is an enzyme of the pyrimidine salvage pathways that metabolizes several cytosine nucleoside analogs used as prodrugs in chemotherapy. We carried out a characterization of the cytidine deaminase 79A>C and 208G>A Single Nucleotide Polymorphisms, in order to highlight their functional role and provide data that could help fine-tune the chemotherapic use of cytosine nucleosides in patients carrying the above mentioned SNPs. The 79A>C SNP results in a K27Q change in a protein region not involved in the catalytic event. The 208G>A SNP produces an alanine to threonine substitution (A70T) within the conserved catalytic domain. Q27 variant is endowed with a greater catalytic efficiency toward the natural substrates and the antileukemic agent cytarabine (Ara-C), when compared to K27 variant. Molecular modeling, protein stability experiments and site-directed mutagenesis suggest that K27 variant may have an increased stability with respect to Q27 due to an ionic interaction between a lysine residue at position 27 and a glutamate residue at position 24. The T70 variant has a lower catalytic efficiency toward the analyzed substrates when compared to the A70 variant, suggesting that patients carrying the 208G>A SNP may have a greater exposure to cytosine based pro drugs, with possible toxicity consequences.

Keywords: 1-β-d-arabino furanosylcytosine or cytarabine; 1-β-d-arabinofuranosyl-5-azacytosine; 2′,2′-difluorodeoxycytidine or gemcitabine; 2′,3′-CdR; 2′,3′-dideoxycytidine; 2′-deoxycytidine; 5′-azadeoxycytidine; 6-aza-CR; 6-azacytidine; Ara-C; Aza-CdR; CDA; CR; CdR; Cytidine deaminase; DTT; EDTA; Genetic polymorphism; IPTG; Kinetic analysis; Molecular modeling; SNP; Single Nucleotide Polymorphisms; Site-directed mutagenesis; THU; cytidine; cytidine deaminase; dFdC; dithiothreitol dithiothreitol; ethylenediaminetetraacetic acid; fazarabine; isopropyl-thio-β-d-galactopyranoside; tetrahydrouridine.

Fig. 1

15% SDS-PAGE of purified CDA variants, and E24Q. (A) Q27/A70, (B) K27/A70, (C) K27/T70, (D) E24Q, st: Bio-Rad low molecular weight standard (97.4 kDa, phosphorylase b; 66.2 kDa, bovine serum albumin; 45.0 kDa, ovalbumin; 31.0 kDa, carbonic anhydrase; 21.5 kDa, soybean trypsin inhibitor; 14.4 kDa, lysozyme).

Fig. 2

Temperature effect on V_max (%) of Q27/A70, K27/A70, K27/T70 functional variants and E24Q mutant enzyme.

Fig. 3

Temperature effect on K_m (van’t Hoff plots) of K27/A70 (A), Q27/A70 (B), K27/T70 (C) and E24Q (D).

Fig. 4

Temperature effect on ln(k_cat/T) (Eyring plots) of K27/A70 (A), Q27/A70 (B), K27/T70 (C) and E24Q (D).

Fig. 5

Effect of sodium hypochlorite and hydrogen peroxide on Q27/A70, K27/A70, K27/T70: (A) time-dependent inhibition by a fixed NaOCl concentration (100 μM); (B) inhibiting effect of NaOCl; (C) time-dependent inhibition by a fixed H₂ O₂ concentration (10 mM); (D) inhibiting effect of H₂ O₂.

Fig. 6

Effect of sodium hypochlorite and hydrogen peroxide on E24Q: (A) time-dependent inhibition by a fixed NaOCl concentration (100 μM); (B) inhibiting effect of sodium hypochlorite; (C) time-dependent inhibition by a fixed H₂ O₂ concentration (10 mM); (D) inhibiting effect of hydrogen peroxide.

Fig. 7

Distances between the nitrogen atom of the side chain of Q27 or K27 and the carboxyl carbon atom of E24 for all the conformations generated through our Monte Carlo conformational searches.

Fig. 8

Most energetically stable structures resulting three Monte Carlo conformational searches conducted to probe the local molecular interactions found in the K27/A70 and Q27/A70 variant. The figures shows the K27/A70 variant (Panel A) shows an ionic interaction between K27 and E24, which is absent in the Q27/A70 variant (Panel B) as well as the K27/A70 variant in which K27 is modified to its chloramine derivative (Panel C).

Fig. 9

Figure of the crystal structure of the Q27/A70 variant of the human CDA (2MQ0, 34) showing the location of Q27 and A70 with respect to the catalytic site. Panel A: view of the CDA tetramer. Panel B: close-up of the catalytic site.