Mammalian Nudt15 hydrolytic and binding activity on methylated guanosine mononucleotides

Abstract

The Nudt15 enzyme of the NUDIX protein family is the subject of extensive study due to its action on thiopurine drugs used in the treatment of cancer and inflammatory diseases. In addition to thiopurines, Nudt15 is enzymatically active in vitro on several nucleotide substrates. It has also been suggested that this enzyme may play a role in 5'RNA turnover by hydrolyzing m⁷GDP, a product of mRNA decapping. However, no detailed studies on this substrate with Nudt15 are available. Here, we analyzed the enzymatic activity of Nudt15 with m⁷GDP, its triphosphate form m⁷GTP, and the trimethylated counterparts (m₃^2,2,7GDP and m₃^2,2,7GTP). Kinetic data revealed a moderate activity of Nudt15 toward these methylated mononucleotides compared to the dGTP substrate. However m⁷GDP and m₃^2,2,7GDP showed a distinct stabilization of Nudt15 upon ligand binding, in the same range as dGTP, and thus these two mononucleotides may be used as leading structures in the design of small molecule binders of Nudt15.

Keywords: Differential scanning fluorimetry; Enzyme kinetics; Methylated mononucleotides; NUDIX family; Nudt15.

Fig. 1

Representative RP-HPLC chromatograms of mNudt15-mediated hydrolysis of guanosine diphosphates (A) and guanosine triphosphates (B). The initial concentration of each indicated substrate was 25 μM, and chromatograms were recorded at the 260 nm wavelength. Analysis of reaction progress after 15 min and 1 h for the indicated compounds are shown. Chromatogram peaks of the analyzed substrate and the corresponding monophosphate product are indicated. (The higher area under the GMP peak after 60 min of reaction (*) could result due to an impurity co-eluting with GMP, as in the additional chromatogram recorded after 2 h of reaction, the area under the GMP peak is comparable to the initial substrate peak area, Supplementary Fig. 3A. Additional peaks (**) seen in the case of m⁷GTP chromatograms correspond to some impurities, as those are not present in the additional chromatograms for this compound, Supplementary Fig. 3B)

Fig. 2

Kinetic curves of mNudt15-mediated hydrolysis of tested mononucleotides. Experimental data points correspond to the initial velocities (presented as nmol of the enzymatically released phosphate per minute) of three independent experiments (± SD), based on the colorimetric assay (as described in Methods). Fitted curves of Michaelis–Menten kinetic model to experimental data points are shown. Solid lines correspond to results obtained at 200 nM mNudt15 concentration, and dotted line to results for trimethylated mononucleotide obtained at 500 nM mNudt15 concentration (kinetic curve for dGTP was omitted for clarity)

Fig. 3

Thermal stabilization of mNudt15 over increasing concentrations of m⁷GDP. (A) Representative DSF melting profiles of mNudt15, RFU—relative fluorescence units. (B) Curves of the first negative derivative of the corresponding melting curves

Fig. 4

Thermal stabilization of mNudt15 as a function of m⁷GDP concentration. ΔTm values data points correspond to of three independent DSF experiments (± SD) and are plotted versus the corresponding concentration of the analyzed compound. Apparent affinity (app.K) was calculated as described by Vivoli et.al. (2014), by fitting to the experimental data to the single site ligand binding model, dashed line (Origin Pro)