Identification of functional domains in Arabidopsis thaliana mRNA decapping enzyme (AtDcp2).
The Arabidopsis thaliana decapping enzyme (AtDcp2) was characterized by bioinformatics analysis and by biochemical studies of the enzyme and mutants produced by recombinant expression. Three functionally significant regions were detected: (i) a highly disordered C-terminal region with a putative PSD-95, Discs-large, ZO-1 (PDZ) domain-binding motif, (ii) a conserved Nudix box constituting the putative active site and (iii) a putative RNA binding domain consisting of the conserved Box B and a preceding loop region. Mutation of the putative PDZ domain-binding motif improved the stability of recombinant AtDcp2 and secondary mutants expressed in Escherichia coli. Such recombinant AtDcp2 specifically hydrolysed capped mRNA to produce 7-methyl GDP and decapped RNA. AtDcp2 activity was Mn(2+)- or Mg(2+)-dependent and was inhibited by the product 7-methyl GDP. Mutation of the conserved glutamate-154 and glutamate-158 in the Nudix box reduced AtDcp2 activity up to 400-fold and showed that AtDcp2 employs the catalytic mechanism conserved amongst Nudix hydrolases. Unlike many Nudix hydrolases, AtDcp2 is refractory to inhibition by fluoride ions. Decapping was dependent on binding to the mRNA moiety rather than to the 7-methyl diguanosine triphosphate cap of the substrate. Mutational analysis of the putative RNA-binding domain confirmed the functional significance of an 11-residue loop region and the conserved Box B.
Gunawardana
Dilantha
D
Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia.
Cheng
Heung-Chin
HC
Gayler
Kenwyn R
KR
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019295:Computational Biology; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005459:Fluorides / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D010720:Phosphines / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion
D029681:Arabidopsis Proteins; D004722:Endoribonucleases; D005459:Fluorides; D018698:Glutamic Acid; D006153:Guanosine Diphosphate; D006639:Histidine; D008239:Lysine; D010720:Phosphines; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C111106:7-methylguanosine 5'-diphosphate; C080938:tris(2-carboxyethyl)phosphine; C033223:polyhistidine; C518494:Dcp2 protein, Arabidopsis