Structural similarities and functional differences clarify evolutionary relationships between tRNA healing enzymes and the myelin enzyme CNPase

Gopinath Muruganandam,Inari Kursula,Arne Raasakka,Petri Kursula,Matti Myllykoski

doi:10.1186/s12858-017-0084-2

Abstract

BackgroundEukaryotic tRNA splicing is an essential process in the transformation of a primary tRNA transcript into a mature functional tRNA molecule. 5′-phosphate ligation involves two steps: a healing reaction catalyzed by polynucleotide kinase (PNK) in association with cyclic phosphodiesterase (CPDase), and a sealing reaction catalyzed by an RNA ligase. The enzymes that catalyze tRNA healing in yeast and higher eukaryotes are homologous to the members of the 2H phosphoesterase superfamily, in particular to the vertebrate myelin enzyme 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase).ResultsWe employed different biophysical and biochemical methods to elucidate the overall structural and functional features of the tRNA healing enzymes yeast Trl1 PNK/CPDase and lancelet PNK/CPDase and compared them with vertebrate CNPase. The yeast and the lancelet enzymes have cyclic phosphodiesterase and polynucleotide kinase activity, while vertebrate CNPase lacks PNK activity. In addition, we also show that the healing enzymes are structurally similar to the vertebrate CNPase by applying synchrotron radiation circular dichroism spectroscopy and small-angle X-ray scattering.ConclusionsWe provide a structural analysis of the tRNA healing enzyme PNK and CPDase domains together. Our results support evolution of vertebrate CNPase from tRNA healing enzymes with a loss of function at its N-terminal PNK-like domain.

Highlights

Eukaryotic transfer ribonucleic acid (tRNA) splicing is an essential process in the transformation of a primary tRNA transcript into a mature functional tRNA molecule. 5′-phosphate ligation involves two steps: a healing reaction catalyzed by polynucleotide kinase (PNK) in association with cyclic phosphodiesterase (CPDase), and a sealing reaction catalyzed by an Ribonucleic acid (RNA) ligase
All constructs contain an N-terminal hexahistidine tag followed by a 3C protease cleavage site and the corresponding PNK/ CPDase domain
A structural model of the PNK/ CPDase proteins (Fig. 9c) can be proposed based on the Conclusions The open elongated conformation of the PNK/CPDases might play a role in RNA substrate binding; this needs to be confirmed by high-resolution structures of the enzymes with bound substrates

Summary

Introduction

Eukaryotic tRNA splicing is an essential process in the transformation of a primary tRNA transcript into a mature functional tRNA molecule. 5′-phosphate ligation involves two steps: a healing reaction catalyzed by polynucleotide kinase (PNK) in association with cyclic phosphodiesterase (CPDase), and a sealing reaction catalyzed by an RNA ligase. The central kinase module of yeast (Saccharomyces cerevisiae) Trl and the N-terminal PNK domain of lancelet PNK/CPDase show resemblance to the bacteriophage T4 PNK and belong to the P-loop phosphotransferase superfamily. They contain the signature Walker A motif (P-loop), which is an NTP-binding site in most NTP-dependent phosphotransferases [8, 11,12,13,14,15]. Initial functional analysis of the kinase module of yeast Trl, using single alanine mutations in the P-loop, revealed that the P-loop motif (401GCGKT405) is a determinant of NTP binding [16]. GTP dependence of the yeast Trl kinase domain has been verified in vitro [17]

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Results

Conclusion