Abstract
The reversible adenine phosphoribosyltransferase enzyme (APRT) is essential for purine homeostasis in prokaryotes and eukaryotes. In humans, APRT (hAPRT) is the only enzyme known to produce AMP in cells from dietary adenine. APRT can also process adenine analogs, which are involved in plant development or neuronal homeostasis. However, the molecular mechanism underlying substrate specificity of APRT and catalysis in both directions of the reaction remains poorly understood. Here we present the crystal structures of hAPRT complexed to three cellular nucleotide analogs (hypoxanthine, IMP, and GMP) that we compare with the phosphate-bound enzyme. We established that binding to hAPRT is substrate shape-specific in the forward reaction, whereas it is base-specific in the reverse reaction. Furthermore, a quantum mechanics/molecular mechanics (QM/MM) analysis suggests that the forward reaction is mainly a nucleophilic substitution of type 2 (SN2) with a mix of SN1-type molecular mechanism. Based on our structural analysis, a magnesium-assisted SN2-type mechanism would be involved in the reverse reaction. These results provide a framework for understanding the molecular mechanism and substrate discrimination in both directions by APRTs. This knowledge can play an instrumental role in the design of inhibitors, such as antiparasitic agents, or adenine-based substrates.
Highlights
The reversible adenine phosphoribosyltransferase enzyme (APRT) is essential for purine homeostasis in prokaryotes and eukaryotes
We left the very unstable PRPP molecules to naturally hydrolyze within PRPP-Mg2ϩ-hAPRT crystal for 1 month
With differential scanning fluorimetry (DSF) analysis, we found no difference in the unfolding temperature when adenine or hypoxanthine were added to the PRPP-Mg2ϩ-hAPRT complex (Table 1)
Summary
The reversible adenine phosphoribosyltransferase enzyme (APRT) is essential for purine homeostasis in prokaryotes and eukaryotes. Two such enzymes in the salvage pathway are adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8) These are enzymes with reversible activities having an ordered sequential bi-bi reaction mechanism [1, 2]. In the forward reaction, APRT synthesizes AMP from adenine (ADE) (Fig. 1), whereas HGPRT generates GMP or IMP from guanine (Gua) or hypoxanthine (Hx), respectively. Both reactions use the co-substrate ␣-D-5-phosphoribosyl-1-pyrophosphate (PRPP) and at least one divalent magnesium ion. To other phosphoribosyltransferase enzymes, both APRT and HGPRT structures are made of a Rossmann fold They include a PRPP-binding motif, a flexible loop, and a hood region (Fig. S1). The means and the standard deviations of the measurements of the unfolding temperatures (Tm) for each protein complex are given
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