Metal requirements and phosphodiesterase activity of tRNase Z enzymes.
The endonuclease tRNase Z from A. thaliana (AthTRZ1) was originally isolated for its tRNA 3' processing activity. Here we show that AthTRZ1 also hydrolyzes the phosphodiester bond in bis(p-nitrophenyl) phosphate (bpNPP) with a kcat of 7.4 s-1 and a KM of 8.5 mM. We analyzed 22 variants of AthTRZ1 with respect to their ability to hydrolyze bpNPP. This mutational mapping identified fourteen variants that lost the ability to hydrolyze bpNPP and seven variants with reduced activity. Surprisingly, a single amino acid change (R252G) resulted in a ten times higher activity compared to the wild type enzyme. tRNase Z enzymes exist in long and short forms. We show here that in contrast to the short tRNase Z enzyme AthTRZ1, the long tRNase Z enzymes do not have bpNPP hydrolysis activity pointing to fundamental differences in substrate cleavage between the two enzyme forms. Furthermore, we determined the metal content of AthTRZ1 and analyzed the metal requirement for bpNPP hydrolysis. AthTRZ1 shows a high affinity for Zn2+ ions; even upon incubation with metal chelators, 0.76 Zn2+ ions are retained per dimer. In contrast to bpNPP hydrolysis, pre-tRNA processing requires additional metal ions, Mn2+ or Mg2+, as Zn2+ ions alone are insufficient.
Molekulare Botanik, Universität Ulm, Albert-Einstein-Allee 11, 89069 Ulm, Germany.
Bettina
B
Späth
Florian
F
Settele
Oliver
O
Schilling
Igor
I
D'Angelo
Andreas
A
Vogel
Ingo
I
Feldmann
Wolfram
W
Meyer-Klaucke
Anita
A
Marchfelder
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D018360:Crystallography, X-Ray; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008345:Manganese / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012343:RNA, Transfer / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*; D001618:beta-Lactamases / Q000378:metabolism
D029681:Arabidopsis Proteins; D029701:Saccharomyces cerevisiae Proteins; D008345:Manganese; D012343:RNA, Transfer; D004722:Endoribonucleases; C512661:tRNase Z, S cerevisiae; C526775:TRZ1 protein, Arabidopsis; D010727:Phosphoric Diester Hydrolases; D001618:beta-Lactamases; D015032:Zinc