Two glutamyl-tRNA reductase activities in Escherichia coli.
delta-Aminolevulinic acid (ALA) is the first committed precursor for tetrapyrrole biosynthesis. ALA formation in Escherichia coli occurs in a tRNA-dependent three-step conversion from glutamate. Glu-tRNA reductase is the key enzyme in this pathway. E. coli K12 contains two Glu-tRNA reductase activities which differ in their molecular weights. Here we describe the purification of one of these enzymes. Four different chromatographic separations yielded a nearly homogeneous protein. Its apparent molecular mass under denaturing (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and nondenaturing conditions (rate zonal sedimentation and gel filtration) is 85,000 +/- 5,000 Da. This indicates a monomeric structure for the active enzyme. Gel filtration and glycerol gradient centrifugation indicate that the other activity has a molecular mass of 45,000 +/- 5,000 Da. In the presence of NADPH both enzyme activities converted E. coli Glu-tRNA(2Glu) to glutamate 1-semialdehyde. Addition of GTP or hemin did not affect the reductase activity. Both enzymes display sequence-specific recognition of tRNA; E. coli Glu-tRNA(2Glu) is a good substrate while the Chlamydomonas reinhardtii, Bacillus subtilis, and Synechocystis Glu-tRNA(Glu) species are poorly recognized.
Jahn
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Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
Michelsen
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Söll
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D000622:Aminolevulinic Acid / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D005971:Glutamates / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006427:Hemin / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D013379:Substrate Specificity
D005971:Glutamates; D009249:NADP; D006427:Hemin; D006160:Guanosine Triphosphate; D000622:Aminolevulinic Acid; D007658:Ketone Oxidoreductases; C059846:glutamyl tRNA dehydrogenase