Abstract
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.
Highlights
8-Aminolevulinic acid, the first committed precursor of the porphyrin moiety of compounds such as hemes and chlorophylls, is formed by two different pathways in nature
Transfer RNA serves in a novelrole in this pathway, namely in the conversion of an amino acid into a low molecular weight metabolite which is not used in protein synthesis
GluTR is not very abundantinthe cell.Only recently hasthe enzyme been purified to near homogeneity from the green alga Chlamydomonas reinhardtii (Chen et al, 1990b). It is a monomer of 130,000 Da, which forms a stable complex with Glu-tRNA and reduces the bound glutamate in the presence of NADPH
Summary
Cloned and found to form an operon with p r f l , the gene of release factor 1 (Li et al, 1989b; Verkamp and Chelm, 1989). The function of the hemA gene product, a 46-kDa protein, is not clear, it may encodea structuralcomponent of the GluTR (Avissar and Beale, 1990)or aregulatory factor that is needed for the expression of the GluTR gene (Avissar and Beale, 1989a). Because of the uncertainty of the assignment of the genetic loci to enzymatic activities we took a different approach and attempted to purify the GluTR activity from E. coli. To our surprise we detected two separable enzyme activities which convert glutamate attached to tRNAG'" into GSA. We describe the purification of one of the activities which has a relative molecular mass of =85,000
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