Expression of catalytically active barley glutamyl tRNAGlu reductase in Escherichia coli as a fusion protein with glutathione S-transferase.
delta-Aminolevulinate in plants, algae, cyanobacteria, and several other bacteria such as Escherichia coli and Bacillus subtilis is synthesized from glutamate by means of a tRNA(Glu) mediated pathway. The enzyme glutamyl tRNA(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(Glu) reductase was expressed in E. coli cells joined at its amino terminal end to Schistosoma japonicum glutathione S-transferase (GST). GST-glutamyl tRNA(Glu) reductase fusion protein and the reductase released from it by thrombin digestion catalyzed the reduction of glutamyl tRNA(Glu) to glutamate 1-semialdehyde. The specific activity of the fusion protein was 120 pmol.micrograms-1.min-1. The fusion protein used tRNA(Glu) from barley chloroplasts preferentially to E. coli tRNA(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.
Vothknecht
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UC
Carlsberg Laboratory, Department of Physiology, Copenhagen-Valby, Denmark.
Kannangara
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CG
von Wettstein
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D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000622:Aminolevulinic Acid / Q000378:metabolism; D001483:Base Sequence; D003580:Cytochromes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006418:Heme / Q000378:metabolism; D006427:Hemin / Q000494:pharmacology; D001467:Hordeum / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry
D003580:Cytochromes; D005971:Glutamates; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid; D006418:Heme; C057794:glutamate-1-semialdehyde; D006427:Hemin; D000622:Aminolevulinic Acid; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase; D005982:Glutathione Transferase