Abstract

The ribokinase superfamily catalyzes the phosphorylation of a vast diversity of substrates, and its members are characterized by the conservation of a common structural fold along with highly conserved sequence motifs responsible for phosphoryl transfer (GXGD) and stabilization of the metal-nucleotide complex (NXXE). Recently, a third motif (HXE) exclusive from ADP-dependent enzymes was identified, with its glutamic acid participating in water-mediated interactions with the metal-nucleotide complex and in stabilization of the ternary complex during catalysis. In this work, we bioinformatically determine that the aspartic acid of another motif (DPV), exclusively found in hydroxyethyl thiazole (THZK), hydroxymethyl pyrimidine (HMPK) and pyridoxal kinases (PLK), is structurally equivalent to the acidic residue in the HXE motif. Moreover, this residue is highly conserved among all ribokinase superfamily members. To determine whether the functional role of the DPV motif is similar to the HXE motif, we employed molecular dynamics simulations using crystal structures of phosphoryl donor substrate-complexed THZK and PLK, showing that its aspartic acid participated in water-mediated or direct interactions with the divalent metal of the metal-nucleotide complex. Lastly, enzyme kinetic assays on human PLK, an enzyme that utilizes zinc, showed that site-directed mutagenesis of the aspartic acid from the DPV motif abolishes the inhibition of this enzyme by increasing free zinc concentrations. Altogether, our results highlight that the DPV and HXE motifs are evolutionary markers of the functional and structural divergence of the ribokinase superfamily and evidence the role of the DPV motif in the interaction with both free and nucleotide-complexed divalent metals in the binding site of these enzymes.

Highlights

  • Enzymes from the ribokinase superfamily can catalyze the phosphorylation of a wide spectrum of substrates, from sugars to nucleosides, and can be found in all three domains of life [1].Their evolutionary relationship is mostly determined by their common Rossmann-like fold, composed of a central beta sheet with at least eight strands in a predominantly parallel disposition surrounded by α-helices [2] and a β-meander subdomain that is located on the C-termini [3].Beyond this conserved fold, these enzymes can be classified into different groups due to structural and functional divergences within this superfamily

  • All members of the superfamily are known for having a strict conservation of catalytic residues required for stabilization of the metal-nucleotide complex (NXXE) [8,9] and for directly participating in the phosphoryl transfer reaction (GXGD) [10]

  • Reasoning that enzymes of the other major groups of the ribokinase superfamily exhibit free divalent metal inhibition [16] or activation [8], we speculated that a motif similar to HXE should be present in the ATP-dependent vitamin and sugar kinases

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Summary

Introduction

Enzymes from the ribokinase superfamily can catalyze the phosphorylation of a wide spectrum of substrates, from sugars to nucleosides, and can be found in all three domains of life [1] Their evolutionary relationship is mostly determined by their common Rossmann-like fold, composed of a central beta sheet with at least eight strands in a predominantly parallel disposition surrounded by α-helices [2] and a β-meander subdomain that is located on the C-termini [3]. Beyond this conserved fold, these enzymes can be classified into different groups due to structural and functional divergences within this superfamily. The ribokinase superfamily members can be classified into three major groups: ATP-dependent sugar kinases (ribokinase family), ATP-dependent vitamin kinases, and ADP-dependent sugar kinases [1]

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