Abstract
Almost all kinases utilize ATP as their phosphate donor, while a few kinases utilize pyrophosphate (PPi) instead. PPi-dependent kinases are often homologous to their ATP-dependent counterparts, but determinants of their different donor specificities remain unclear. We identify a PPi-dependent member of the ribokinase family, which differs from known PPi-dependent kinases, and elucidate its PPi-binding mode based on the crystal structures. Structural comparison and sequence alignment reveal five important residues: three basic residues specifically recognizing PPi and two large hydrophobic residues occluding a part of the ATP-binding pocket. Two of the three basic residues adapt a conserved motif of the ribokinase family for the PPi binding. Using these five key residues as a signature pattern, we discover additional PPi-specific members of the ribokinase family, and thus conclude that these residues are the determinants of PPi-specific binding. Introduction of these residues may enable transformation of ATP-dependent ribokinase family members into PPi-dependent enzymes.
Highlights
Almost all kinases utilize ATP as their phosphate donor, while a few kinases utilize pyrophosphate (PPi) instead
Structural comparison and sequence alignment with ATP-dependent or ADP-dependent members of the ribokinase family reveal the importance of five residues: two large hydrophobic residues occluding a part of the ATP-binding pocket and three basic residues involved in PPi recognition
A PPi-dependent kinase belonging to the ribokinase family was identified based on structural similarity to a myo-inositol 3-kinase from the hyperthermophilic archaeon Thermococcus kodakarensis (MI3K_TK), which is an ATP-dependent member of the ribokinase family[19,20]
Summary
Almost all kinases utilize ATP as their phosphate donor, while a few kinases utilize pyrophosphate (PPi) instead. We identify a PPi-dependent member of the ribokinase family, which differs from known PPi-dependent kinases, and elucidate its PPi-binding mode based on the crystal structures. Structural comparison and sequence alignment reveal five important residues: three basic residues recognizing PPi and two large hydrophobic residues occluding a part of the ATP-binding pocket. PPi-ACK from E. histolytica is thought to primarily produce PPi and acetate from Pi and acetyl phosphate under physiological conditions because the kcat value of the PPiproducing reaction is 1000-fold higher than that of the PPiconsuming reaction[7]. Structural comparison and sequence alignment with ATP-dependent or ADP-dependent members of the ribokinase family reveal the importance of five residues: two large hydrophobic residues occluding a part of the ATP-binding pocket and three basic residues involved in PPi recognition. The five residues are used collectively as a signature pattern and enable us to newly identify PPi-specific members of the ribokinase family
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