A Multi-enzyme Cascade for the Biosynthesis of AICA Ribonucleoside Di- and Triphosphate

Abstract

AICA (5'-aminoimidazole-4-carboxamide) ribonucleotides with different phosphorylation levels are the pharmaceutically active metabolites of AICA nucleoside-based drugs. The chemical synthesis of AICA ribonucleotides with defined phosphorylation is challenging and expensive. In this study, we describe two enzymatic cascades to synthesize AICA derivatives with defined phosphorylation levels from the corresponding nucleobase and the co-substrate phosphoribosyl pyrophosphate. The cascades are composed of an adenine phosphoribosyltransferase from Escherichia coli (EcAPT) and different polyphosphate kinases: polyphosphate kinase from Acinetobacter johnsonii (AjPPK), and polyphosphate kinase from Meiothermus ruber (MrPPK). The role of the EcAPT is to bind the nucleobase to the sugar moiety, while the kinases are responsible for further phosphorylation of the nucleotide to produce the desired phosphorylated AICA ribonucleotide. The selected enzymes were characterized, and conditions were established for two enzymatic cascades. The diphosphorylated AICA ribonucleotide derivative ZDP, synthesized from the cascade EcAPT/AjPPK, was produced with a conversion up to 91 %. The EcAPT/MrPPK cascade yielded ZTP with conversion up to 65 % with ZDP as a side product.

Keywords: adenine phosphoribosyltransferase; enzymatic nucleotide synthesis; enzyme cascades; nucleotide analogues; polyphosphate kinases.

Figure 1

Desired cascades for synthesizing 5‐aminoimidazole‐4‐carboxamide ribonucleotide mono‐, di‐ and tri‐phosphate (ZMP, ZDP, and ZTP). The adenine phosphoribosyltransferase (EcAPT) catalyzes the coupling of the base AICA to the ribose moiety (PRPP) to form ZMP. A combination of the EcAPT with different polyphosphate kinases shall then be used to create cascades for the biosynthesis of ZDP (AjPPK) and ZTP (MrPPK).

Figure 2

Influence of pH value (A) and reaction temperature (B) on the activity of the enzymes EcAPT, AjPPK, and MrPPK. For the pH experiments, the reaction temperature was kept constant at 40 °C for EcAPT and MrPPK and at 30 °C for AjPPK. All experiments were performed in tris‐HCl buffer, set to the respective pH Value. For the temperature experiments, the pH was maintained at 8.0 for all enzymes. The error bars indicate the standard deviation from the three independent replicates.

Figure 3

AICA nucleotide concentrations over the reaction time in the cascades EcAPT/AjPPK (A) and EcAPT/MrPPK (B). Enzyme concentrations: A: EcAPT 1 μmol L⁻¹, AjPPK 0.05 μmol L⁻¹. B: EcAPT 1 μmol L⁻¹, MrPPK 10 μmol L⁻¹. Substrate concentrations: 10 mmol L⁻¹ AICA, 2 mmol L⁻¹ PRPP. The error bars indicate the standard deviation from the three independent replicates.

Figure 4

Effect of variation of enzyme concentrations. A. EcAPT/AjPPK cascade with EcAPT concentrations between 1–5 μmol L⁻¹ and AjPPK concentrations between 0.05–0.5 μmol L⁻¹. The reactions were performed for 2 h at 30 °C. B. EcAPT/MrPPK cascade with EcAPT concentrations between 1–5 μmol L⁻¹ and MrPPK concentrations between 10–40 μmol L⁻¹. Substrate concentrations: 10 mmol L⁻¹ AICA, 2 mmol L⁻¹ PRPP. The reactions were performed for 1 h at 40 °C. The error bars indicate the standard deviation from the three independent replicates.

Figure 5

Effect of sodium polyphosphate concentrations (5–50 mmol L⁻¹) on the cascades EcAPT/AjPPK (A) and EcAPT/MrPPK (B). Enzyme concentrations: A: EcAPT1, MrPPK 0.1 μmol L⁻¹; B: EcAPT 1, MrPPK 10 μmol L⁻¹. Substrate concentrations: 10 mmol L⁻¹ AICA, 2 mmol L⁻¹ PRPP. The reactions were performed for 2 h at 30 °C (A) and 1 h at 40 °C (B), respectively. The error bars indicate the standard deviation from the three independent replicates.

Figure 6

EcAPT/AjPPK/MrPPK cascade with EcAPT concentration 1 μmol L⁻¹, AjPPK concentrations between 0.02–0.1 μmol L⁻¹, and MrPPK concentrations between 2–20 μmol L⁻¹ with 25 mmol L⁻¹ sodium polyphosphate, 10 mmol L⁻¹ AICA and 2 mmol L⁻¹ PRPP. The reaction was performed for 2 h at 30 °C The error bars indicate the standard deviation from the three independent replicates.