Abstract
The 2H phosphoesterase family contains enzymes with two His-X-Ser/Thr motifs in the active site. 2H enzymes are found in all kingdoms of life, sharing little sequence identity despite the conserved overall fold and active site. For many 2H enzymes, the physiological function is unknown. Here, we studied the structure of the 2H family member LigT from Escherichia coli both in the apo form and complexed with different active-site ligands, including ATP, 2′-AMP, 3′-AMP, phosphate, and NADP+. Comparisons to the well-characterized vertebrate myelin enzyme 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) highlight specific features of the catalytic cycle and substrate recognition in both enzymes. The role played by the helix α7, unique to CNPases within the 2H family, is apparently taken over by Arg130 in the bacterial enzyme. Other residues and loops lining the active site groove are likely to be important for RNA substrate binding. We visualized conformational changes related to ligand binding, as well as the position of the nucleophilic water molecule. We also present a low-resolution model of E. coli LigT bound to tRNA in solution, and provide a model for RNA binding by LigT, involving flexible loops lining the active site cavity. Taken together, our results both aid in understanding the common features of 2H family enzymes and help highlight the distinct features in the 2H family members, which must result in different reaction mechanisms. Unique aspects in different 2H family members can be observed in ligand recognition and binding, and in the coordination of the nucleophilic water molecule and the reactive phosphate moiety.
Highlights
The 2H phosphoesterase superfamily is an ancient group of proteins and protein domains characterized by a common fold and a few conserved active site residues [1,2]
Various catalytic activities have been assigned for the 2H enzymes: hydrolysis of a 20,30-cyclic phosphate, in either nucleotides or 30-ends of RNA molecules, into 20-phosphate [3,4,5], hydrolysis of ADPribose 100,200-cyclic phosphates into ADP-ribose 100-phosphate [6,7,8], generation and/or cleavage of 20-50-linkages between nucleotides or RNA molecules, such as tRNA halves [3,9,10,11,12,13], and 30-50 exonucleolytic removal of terminal uridine nucleotides from snRNA with the simultaneous generation of 20,30-cyclic phosphates at the terminus [14]
The structure of the putative tRNA ligase LigT from E. coli was determined by X-ray crystallography in the apo form
Summary
The 2H phosphoesterase superfamily is an ancient group of proteins and protein domains characterized by a common fold and a few conserved active site residues [1,2]. E. coli LigT was crystallized, and its structure was refined in complex with the in vitro reaction product 20-AMP [22]. These water molecules coordinate the substrate/product throughout the reaction in CNPase [17,18], and they are likely to play a similar role in LigT and other 2H phosphoesterases.
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