Expression of glutamyl-tRNA reductase in Escherichia coli.
The biosynthesis of the hemes, chlorophylls, corrins and other tetrapyrroles begins with the synthesis of 5-aminolevulinic acid (ALA). The pathway is highly conserved except for the synthesis of ALA which is derived from glycine and succinyl CoA (C4) in most eukaryotes and from glutamate (C5) in most bacteria and in green plants. In C5, glutamyl-tRNA synthetase (GTS) converts glutamate to glutamyl-tRNA (glu-tRNA), which is reduced by glutamyl-tRNA reductase (GTR) to glutamyl-1-semialdehyde (GSA), which is converted by aminotransferase (GSA-AT) to ALA. Since GTS is also involved in protein synthesis and GSA can be converted to ALA non-enzymatically, it is highly probable that control of ALA synthesis and thus of the whole pathway resides in the GTR step. In Escherichia coli, GTR is the gene product of hemA. BL21(DE3), a protease-deficient strain which contains the T7 RNA polymerase gene in front of a lac promoter, was transformed with a pET14b-based vector, pWC01, harboring hemA in front of a T7 promoter and ORF1 which is transcribed in the opposite direction. The transformed strain, WC1201, secreted ALA and porphyrins into the medium. Induction of expression of hemA by WC1201 was optimized for concentration of inducer (IPTG, 5 mM), temperature (37 degrees C), presence of betaine and sorbitol (no change) and time of induction (2h). GTR was observable as a 46 kDa band by Brilliant blue G staining of SDS-PAGE gels. Sonicates of the induction mixture exhibited strong ALA synthesis activity which was enhanced by tRNAglu. Most of the activity was in the supernatant of the sonicate indicating that GTR is a soluble enzyme. The induced strain had more GTS activity than the uninduced strain which had more GTS activity than its parent wild-type strain. Autoradiography on native gradient PAGE showed that GTR expressed in vivo by induction of WC1201 had a molecular weight of approx. 117 kDa. Gel filtration of the induced sonicate showed a peak of enzymatic activity at about 126 kDa. When pET14b- or pUC19-based plasmids harboring hemA and ORF1, or importantly, a pUC19-based plasmid harboring only hemA and not ORF1, were expressed in an in vitro transcription-translation system, native gradient PAGE showed a product with a molecular weight of approximately 175 kDA. This expression was higher in the presence of tRNAglu. When the 117 kDa and 175 kDa proteins were excised from their native gels respectively, and run on SDS PAGE, autoradiography showed bands at 46 kDa. We conclude that GTR is present in both high molecular weight species. Since overexpression of hemA from pET14b-based plasmids is associated with increased glutamyl-tRNA synthetase activity, the 175 kDa species may represent different complexes of GTR, GTS and glutamyl-tRNA as observed in Chlamydomonas and the 117-126 kDa species may be an dimer of GTR associated with glu-tRNA or a complex of GTR, GTS and glu-tRNA. These possibilities are being investigated.
Chen
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Department of Biology, City College of New York, City University of New York, NY 10031, USA.
Wright
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Li
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Cosloy
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Russell
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Lee
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D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000622:Aminolevulinic Acid / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D008970:Molecular Weight; D016366:Open Reading Frames / Q000235:genetics; D012352:RNA, Transfer, Glu / Q000378:metabolism; D011993:Recombinant Fusion Proteins
D012352:RNA, Transfer, Glu; D011993:Recombinant Fusion Proteins; D000622:Aminolevulinic Acid; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase