Expression of catalytically active barley glutamyl tRNAGlu reductase in Escherichia coli as a fusion protein with glutathione S-transferase

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Date: Aug. 20, 1996
Publisher: National Academy of Sciences
Document Type: Article
Length: 202 words

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Abstract :

[Delta]-Aminolevulinate in plants, algae, cyanobacteria, and several other bacteria such as Escherichia coil and Bacillus subtilis is synthesized from glutamate by means of a [tRNA.sup.Glu] mediated pathway. The enzyme glutamyl [tRNA.sup.Glu] reductase catalyzes the second step in this pathway, the reduction of tRNA bound glutamate to give glutamate 1-semialdehyde. The hemA gene from barley encoding the glutamyl [tRNA.sup.Glu] reductase was expressed in E. coli cells joined at its amino terminal end to Schistosoma japonicum glutathione S-transferase (GST). GST-glutamyl [tRNA.sup.Glu] reductase fusion protein and the reductase released from it by thrombin digestion catalyzed the reduction of glutamyl [tRNA[not equal to]Glu] to glutamate 1-semialdehyde. The specific activity of the fusion protein was 120 pmol[center dot][[[micro]gram].sup.1][center dot][min.sup.-1]. The fusion protein used [tRNA.sup.Glu] from barley chloroplasts preferentially to E. coli [tRNA.sup.Glu] and its activity was inhibited by hemin. It migrated as an 82-kDa polypeptide with SDS/PAGE and eluted with an apparent molecular mass of 450 kDa from Superose 12. After removal of the GST by thrombin, the protein migrated as an [approximately equal to]60-kDa polypeptide with SDS/PAGE, whereas gel filtration on Superose 12 yielded an apparent molecule mass of 250 kDa. Isolated fusion protein contained heme, which could be reduced by NADPH and oxidized by air.

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Gale Document Number: GALE|A18701728