Structures of Yeast Apa2 Reveal Catalytic Insights into a Canonical Ap4A Phosphorylase of the Histidine Triad Superfamily

The homeostasis of intracellular diadenosine 5',5 '''-P-1,P-4-tetraphosphate (Ap(4)A) in the yeast Saccharomyces cerevisiae is maintained by two 60% sequence-identical paralogs of Ap(4)A phosphorylases (Apa1 and Apa2). Enzymatic assays show that, compared to Apa1, Apa2 has a relatively higher phosphorylase activity towards Ap(3)A (5',5 '''-P-1,P-3-tetraphosphate), Ap(4)A, and Ap(5)A (5',5 '''-P-1,P-3-tetraphosphate), and Ap(4)A is the favorable substrate for both enzymes. To decipher the catalytic insights, we determined the crystal structures of Apa2 in the apo-, AMP-, and Ap(4)A-complexed forms at 2.30, 2.80, and 2.70 angstrom resolution, respectively. Apa2 is an alpha/beta protein with a core domain of a twisted eight-stranded antiparallel beta-sheet flanked by several alpha-helices, similar to the galactose-1-phosphate uridylyltransferase (GalT) members of the histidine triad (HIT) superfamily. However, a unique auxiliary domain enables an individual Apa2 monomer to possess an intact substrate-binding cleft, which is distinct from previously reported dimeric GaIT proteins. This cleft is perfectly complementary to the favorable substrate Ap(4)A, the AMP and ATP moieties of which are perpendicular to each other, leaving the a-phosphate group exposed at the sharp turn against the catalytic residue His161. Structural comparisons combined with site-directed mutagenesis and activity assays enable us to define the key residues for catalysis. Furthermore, multiple-sequence alignment reveals that Apa2 and homologs represent canonical Ap(4)A phosphorylases, which could be grouped as a unique branch in the GaIT family. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved.