Structural And Enzymatic Characterization Of Streptococcal Ap_nA Hydrolase SapH And Yeast Ap₄A Phosphorylase Apa2

Diadenosine polyphosphates (ApnAs) are a class of nucleotide derivatives distributed in all types of organisms. They are mainly produced by aminoacyl-tRNA synthetases as by-products during protein synthesis. They have emerged as intracellular and extracellular signal molecules implicated in the maintenance and regulation of vital cellular functions and become considered as second messengers. The accumulation of these molecules could inhibit the activity of several key enzymes such as adenylate kinases and protein kinases. Thus ApnAs metabolism, especially the degradation of these molecules is crucial for maintaining their intracellular homoestasis.Specific enzymes for degrading ApnAs have been found in all kingdoms of life, and they could be generally classified into three categories, Nudix (nucleoside diphosphate linked to x) superfamily, HIT (histidine triad) superfamily and MPP (metallophosphodiesterase) superfamily. We selected Streptococcus pneumonia R6ApnA hydrolase Sprl479/SapH and Saccharomyces cerevisiae Ap4A phosphorylase Apa2as our targets. They belong to MPP and HIT superfamily, respectively, which are considered to be important for the homoestasis of ApnAs.Sprl479from S. pneumoniae R6is a33-kDa hypothetical protein of unknown function. Here, we determined the crystal structures of its apo-form at1.90A, and complex forms with inorganic phosphate and AMP at2.30A and2.20A, respectively. The core structure of Sprl479adopts a four-layered α-β-β-α sandwich fold, with Fe3+and Mn2+coordinated at the binuclear center of the active site (similar to metallophosphoesterases). Enzymatic assays show that in addition to phosphodiesterase activity towards bis-(p-nitrophenyl) phosphate, Spr1479has hydrolase activity towards ApnA and ATP. Residues that coordinate with the two metals are indispensable for both activities. By contrast, the Strepttococci-specific residue Trp67, which binds to phosphate in the two complex structures, is indispensable for the ATP/ApnA hydrolase activity only. Moreover, the AMP-binding pocket is exclusively conserved in all Streptococci. Therefore, we named the protein SapH, for Streptococcal ATP/ApnA and phosphodiester hydrolase.The degradation of Ap4A in S. cerevisiae is mainly catalyzed by two Ap4A phosphorylases (Apal and Apa2) with60%sequence identity. They could reversely catalyze the degradation of Ap4A into ADP and ATP. The disruption of apal alone, apa2alone, and both genes did not affect cell viability, whereas the absence of both genes could increase the intracellular concentration of Ap4A dramatically. Here we report the crystal structures of Apa2apo-form and Ap4A-complex form at2.3and2.6A, respectively. Apa2adopts an α/β fold, with the core structure of seven-stranded β-sheet resembling the GalT (galactose-1-phosphate uridylyltransferase) members of HIT superfamily. The additional sub-domain which is unique in Apa2forms a part of Ap4A-binding pocket. The AMP and ATP moiety of Ap4A is perpendicular to each other, thus exposing the a-phosphate group to the catalytic residue His161for nucleophilic attack. Similar to other GalT members, Apa2also adopts a ping-pong catalytic mechanism and forms the nucleotide-His intermediate. Enzymatic assays show that the relative activity of Apal towards Ap4A is only about one seventh of Apa2, which mainly results from the substitution of Leu67of Apal to Phe68of Apa2. In addition to Ap4A phosphorylase activity (kcat/Km of21.0s-1μM-1), Apa2also shows activity towards Ap3A and Ap5A, with the relative activity of one twelfth and one third to that of Ap4A, respectively. Ap4A is the favorable substrate because the binding pocket is perfectly complementary to a molecule of Ap4A. Multiple-sequence alignment reveals that Apa2evolves independently and represents a new branch of GalT family. This is also the first structure of a classic Ap4A phosphorylase in HIT superfamily.