Physical and kinetic interactions between glutamyl-tRNA reductase and glutamate-1-semialdehyde aminotransferase of Chlamydomonas reinhardtii.
In plants, algae, and most bacteria, the heme and chlorophyll precursor 5-aminolevulinic acid (ALA) is formed from glutamate in a three-step process. First, glutamate is ligated to its cognate tRNA by glutamyl-tRNA synthetase. Activated glutamate is then converted to a glutamate 1-semialdehyde (GSA) by glutamyl-tRNA reductase (GTR) in an NADPH-dependent reaction. Subsequently, GSA is rearranged to ALA by glutamate-1-semialdehyde aminotransferase (GSAT). The intermediate GSA is highly unstable under physiological conditions. We have used purified recombinant GTR and GSAT from the unicellular alga Chlamydomonas reinhardtii to show that GTR and GSAT form a physical and functional complex that allows channeling of GSA between the enzymes. Co-immunoprecipitation and sucrose gradient ultracentrifugation results indicate that recombinant GTR and GSAT enzymes specifically interact. In vivo cross-linking results support the in vitro results and demonstrate that GTR and GSAT are components of a high molecular mass complex in C. reinhardtii cells. In a coupled enzyme assay containing GTR and wild-type GSAT, addition of inactive mutant GSAT inhibited ALA formation from glutamyl-tRNA. Mutant GSAT did not inhibit ALA formation from GSA by wild-type GSAT. These results suggest that there is competition between wild-type and mutant GSAT for binding to GTR and channeling GSA from GTR to GSAT. Further evidence supporting kinetic interaction of GTR and GSAT is the observation that both wild-type and mutant GSAT stimulate glutamyl-tRNA-dependent NADPH oxidation by GTR.
Nogaj
Luiza A
LA
Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA.
Beale
Samuel I
SI
D000445:Aldehyde Oxidoreductases / Q000737:chemistry*; D000818:Animals; D001667:Binding, Competitive; D002384:Catalysis; D002499:Centrifugation, Density Gradient; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D047468:Immunoprecipitation; D019751:Intramolecular Transferases / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013395:Sucrose / Q000737:chemistry / Q000494:pharmacology; D013997:Time Factors
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D012346:RNA, Transfer, Amino Acyl; D011994:Recombinant Proteins; D009249:NADP; D013395:Sucrose; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase; D010100:Oxygen