Purification and functional characterization of the Glu-tRNA(Gln) amidotransferase from Chlamydomonas reinhardtii.

D Jahn,Y C Kim,Y Ishino,M W Chen,D Söll

doi:10.1016/s0021-9258(19)39038-6

Abstract

The formation of glutaminyl-tRNA (Gln-tRNA) in Bacilli, chloroplasts, and mitochondria occurs in a two-step reaction. This involves misacylation of tRNA(Gln) with glutamate by glutamyl-tRNA synthetase and subsequent amidation of Glu-tRNA(Gln) to the correctly acylated Gln-tRNA(Gln) by a specific amidotransferase (Schön, A., Kannangara, C. G., Gough, S., and Söll, D. (1988) Nature 331, 187-190). Here we demonstrate the existence of this pathway in green algae and describe the purification of the Glu-tRNA(Gln) amidotransferase from Chlamydomonas reinhardtii. The purified enzyme showed an Mr of approximately 120,000 when analyzed by glycerol gradient sedimentation and gel filtration. An apparent Mr of 63,000 of the denatured protein was demonstrated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. This indicates that the enzyme possesses an alpha 2 structure. The substrate for the purified enzyme is Glu-tRNA(Gln) but not Glu-tRNA(Glu). The enzyme requires ATP, Mg2+, and an amide donor for the conversion. Acceptable amide donors are glutamine, asparagine, and ammonia. Blocking of the glutamine-dependent reaction by alkylation of the protein with 6-diazo-5-oxonorleucine did not inhibit the ammonia-dependent reaction, suggesting that the enzyme has separate glutamine and ammonia binding sites. As suggested by Wilcox (Wilcox, M. (1969) Eur. J. Biochem. 11, 405-412) the amidation reaction may involve glutamyl-phosphate formation, since ATP is cleaved to ADP when the enzyme is incubated with Glu-tRNA(Gln) and ATP. In common with other glutamine amidotransferases, the enzyme also possesses low glutaminase activity. The purified Glu-tRNA(Gln) amidotransferase forms a stable complex with Glu-tRNA(Gln) in the presence of ATP and Mg2+ but in the absence of the amide donor as determined by gradient centrifugation.

Highlights

Since it is known that glutamine synthetase can carry out at low efficiency the conversion of GlutRNAG’” + Gln-tRNA”” (Strauch et al, 1988), we assayed for this enzyme (Lipmann and Tuttle, 1945)
Amide Donor-When we examined the ability of the puri- Earlier experiments (Wilcox, 1969) provided evidence that fied AdT to use different amide donors we found that a ATP is required in the reaction to activate the y-carboxyl number of glutamine analogs substituted for glutamine
Since the presence of a functional glutaminyl-tRNA synthetase could not be demonstrated in these organisms and organelles, this pathway provides the sole source of glutamine for protein biosynthesis

Summary

B Present address

Gln-tRNAG’” by amidation of a misacylated Glu-tRNAG1”. This amidation reaction is catalyzed by a tRNA-specific glutamine amidotransferase (see Fig. 1). Amino acid sequence comparison of these enzymes obtained with the results of enzymatic studies provided evidence for two distinct types of glutamine amidotransferases designated as the trpG- andpuFF-type subfamilies (Weng and Zalkin, 1987; Mei and Zalkin; 1989). The Glu-tRNA”‘” amidotransferase is unique among this class of enzymes in its use of Glu-tRNA as amide acceptor. As there is little known about the structure and properties of these tRNA-dependent enzymes, we decided to purify the Glu-tRNA”‘” amidotransferase from Chlamydomonas reinhardtii.

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DISCUSSION