Role of glutamate 64 in the activation of the prodrug 5-fluorocytosine by yeast cytosine deaminase

Abstract

Yeast cytosine deaminase (yCD) catalyzes the hydrolytic deamination of cytosine to uracil as well as the deamination of the prodrug 5-fluorocytosine (5FC) to the anticancer drug 5-fluorouracil. In this study, the role of Glu64 in the activation of the prodrug 5FC was investigated by site-directed mutagenesis, biochemical, nuclear magnetic resonance (NMR), and computational studies. Steady-state kinetics studies showed that the mutation of Glu64 causes a dramatic decrease in k(cat) and a dramatic increase in K(m), indicating Glu64 is important for both binding and catalysis in the activation of 5FC. (19)F NMR experiments showed that binding of the inhibitor 5-fluoro-1H-pyrimidin-2-one (5FPy) to the wild-type yCD causes an upfield shift, indicating that the bound inhibitor is in the hydrated form, mimicking the transition state or the tetrahedral intermediate in the activation of 5FC. However, binding of 5FPy to the E64A mutant enzyme causes a downfield shift, indicating that the bound 5FPy remains in an unhydrated form in the complex with the mutant enzyme. (1)H and (15)N NMR analysis revealed trans-hydrogen bond D/H isotope effects on the hydrogen of the amide of Glu64, indicating that the carboxylate of Glu64 forms two hydrogen bonds with the hydrated 5FPy. ONIOM calculations showed that the wild-type yCD complex with the hydrated form of the inhibitor 1H-pyrimidin-2-one is more stable than the initial binding complex, and in contrast, with the E64A mutant enzyme, the hydrated inhibitor is no longer favored and the conversion has a higher activation energy, as well. The hydrated inhibitor is stabilized in the wild-type yCD by two hydrogen bonds between it and the carboxylate of Glu64 as revealed by (1)H and (15)N NMR analysis. To explore the functional role of Glu64 in catalysis, we investigated the deamination of cytosine catalyzed by the E64A mutant by ONIOM calculations. The results showed that without the assistance of Glu64, both proton transfers before and after the formation of the tetrahedral reaction intermediate become partially rate-limiting steps. The results of the experimental and computational studies together indicate that Glu64 plays a critical role in both the binding and the chemical transformation in the conversion of the prodrug 5FC to the anticancer drug 5-fluorouracil.

Figure 1

Interactions of the transition state analogue DHP with yCD. The drawing is based on the 1.14 Å-resolution crystal structure of yCD in complex with DHP (PDB code 1P6O) (5). Hydrogen atoms were added using InsightII. The zinc atom (green) is coordinated with His62, Cys91, Cys94 and the 4-hydroxyl group of DHP.

Figure 2

¹⁹F-NMR spectra of the wild-type yCD-5FPy complex and the E64A-5FPy complex. All samples were in a D₂O buffer containing 100 mM potassium phosphate, pH 7.5. (A) Spectrum of 5FPy (4.0 mM) in the absence of the wild-type yCD or E64A; (B) spectrum of 5FPy (3.0 mM) in the presence of the wild-type yCD (1.5 mM); (C) spectrum of 5FPy (3.0 mM) in the presence of E64A (1.5 mM).

Figure 3

Region of the ¹⁵N ¹H IS-TROSY spectrum of Glu64 backbone amide resonance. The inset is the schematic diagram of H-bonding between yCD and 5F-DHP derived from the 1.14 Å resolution crystal structure (PDB code: 1P6O) (5). Lengths (Å) of H-bonds are indicated.

Figure 4

Proposed reaction pathway from Py to DHP catalyzed by the wild type yCD.

Figure 5

ONIOM optimized structures for the conversion of Py to DHP by the wild type yCD. (1) complex 1, (2) TS12 between complex 1 and 2, (3) complex 2, (4) TS23 between complex 2 and 3, (5) complex 3, (6) TS34 between complex 3 and 4, (7) complex 4, (8) TS45 between complex 4 and 5, (9) complex 5.

Figure 6

Schematic E^ONIOM and E(High,model) energy profile for the conversion of Py to DHP catalyzed by the wild-type yCD.

Figure 7

Proposed reaction pathway from Py to DHP catalyzed by the E64A mutant enzyme.

Figure 8

ONIOM optimized structures for the conversion of Py to DHP by the E64A mutant enzyme. (1) complex 6, (2) TS67 between complex 6 and 7, (3) complex 7.

Figure 9

Schematic E^ONIOM and E(High,model) energy profile for the conversion from Py to DHP catalyzed by the E64A mutant enzyme.

Figure 10

Conversion of cytosine to the zinc-coordinated uracil catalyzed by the E64A mutant enzyme.

Figure 11

ONIOM optimized structures for the conversion from cytosine to the zinc-coordinated uracil. (1) complex 8, (2) TS89 between complex 8 and 9, (3) complex 9, (4) TS910 between complex 9 and 10 and (5) complex 10.

Figure 12

Schematic E^ONIOM and E(High,model) energy profile for the conversion from cytosine to uracil catalyzed by the E64A mutant enzyme.