Structural Insights into the Forward and Reverse Enzymatic Reactions in Human Adenine Phosphoribosyltransferase

Abstract

Phosphoribosyltransferases catalyze the displacement of a PRPP α-1'-pyrophosphate to a nitrogen-containing nucleobase. How they control the balance of substrates/products binding and activities is poorly understood. Here, we investigated the human adenine phosphoribosyltransferase (hAPRT) that produces AMP in the purine salvage pathway. We show that a single oxygen atom from the Tyr105 side chain is responsible for selecting the active conformation of the 12 amino acid long catalytic loop. Using in vitro, cellular, and in crystallo approaches, we demonstrated that Tyr105 is key for the fine-tuning of the kinetic activity efficiencies of the forward and reverse reactions. Together, our results reveal an evolutionary pressure on the strictly conserved Tyr105 and on the dynamic motion of the flexible loop in phosphoribosyltransferases that is essential for purine biosynthesis in cells. These data also provide the framework for designing novel adenine derivatives that could modulate, through hAPRT, diseases-involved cellular pathways.

Phytohormone metabolism in human cells: Cytokinins are taken up and interconverted in HeLa cell culture

“…APRT has been documented in mammalian systems where it is capable of converting purine bases into their respective monophosphates . Specifically, APRT is involved in the purine nucleotide salvage pathway, where it catalyzes the addition of a phosphoribosyl group from the phosphoribosyl pyrophosphate (PRPP) molecule to adenine, thus forming AMP and releasing pyrophosphate (PPi) . Therefore, this study supports previous findings that APRT is active in mammalian cells, facilitating conversions from the nucleotide form of CKs directly into the FB form.…”

“…54,55,59 Specifically, APRT is involved in the purine nucleotide salvage pathway, where it catalyzes the addition of a phosphoribosyl group from the phosphoribosyl pyrophosphate (PRPP) molecule to adenine, thus forming AMP and releasing pyrophosphate (PPi). 60 Therefore, this study supports previous findings that APRT is active in mammalian cells, facilitating conversions from the nucleotide form of CKs directly into the FB form. (either mono-, di-, or tri-) phosphate; riboside (BARP), benzyladenine riboside (BAR); free base (FB), and benzyladenine (BA) detected in the treated HeLa culture supernatants and the proposed enzymes responsible for the interconversions between cytokinin (CK) structural forms.…”

Structural basis for substrate selectivity and nucleophilic substitution mechanisms in human adenine phosphoribosyltransferase catalyzed reaction

“…Four crystallographic structures of human APRT in complex with either phosphate ion, Hx-PRPP-Mg 2ϩ , IMP, or GMP were obtained. Our results, compared with our recent ADE-PRPP-Mg 2ϩ -, AMP-, and AMP-PP i -Mg 2ϩ -hAPRT structures (6), show that hAPRT follows mainly an S N 2-type mechanism in both directions of the reactions, with some S N 1-type character, and discriminates substrates according to their molecular shapes, chemical composition, and a loop closure over the active site.…”

“…The last seems to provide purine specificity, either ADE or Hx and Gua, in APRT or HGPRT, respectively (3,4). Furthermore, the flexible loop is very dynamic (5), and we recently showed that a conserved tyrosine within the flexible loop of human APRT facilitates the forward reaction and is essential for cell growth (6).…”

Canis familiaris tissues are characterized by different profiles of cytokinins typical of the tRNA degradation pathway

“…As documented in plant systems, FBs may be used as substrates for adenine phosphoribosyltransferases (15). In humans, adenine phosphoribosyltransferases are involved in the nucleotide salvage pathway responsible for AMP synthesis from adenine and a-D-5-phosphoribosyl-1-pyrpophosphate (25).…”