Physical and Kinetic Interactions between Glutamyl-tRNA Reductase and Glutamate-1-semialdehyde Aminotransferase of Chlamydomonas reinhardtii

Luiza A Nogaj,Samuel I Beale

doi:10.1074/jbc.m502483200

Abstract

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.

Highlights

5-Aminolevulinic acid (ALA)1 is the earliest universal precursor in the tetrapyrrole biosynthesis pathway [1, 2]
Determination of glutamyl-tRNA reductase (GTR) Activity by aminolevulinic acid (ALA) Formation—GTR activity was assayed as ALA formation from glutamyl-tRNA in a coupled enzyme assay carried out in 96-well microtiter plates in 0.1 ml of assay buffer supplemented with 40 ␮g of purified recombinant C. reinhardtii glutamate-1-semialdehyde aminotransferase (GSAT), 80 units of E. coli aminoacyl-tRNA synthetases (Sigma), 0.01 units of E. coli tRNAGlu (Sigma), 5 mM ATP, 5 mM levulinic acid, 1 mM NADPH, 1 mM glutamate, 1 mM dithiothreitol, and 20 ␮M pyridoxal-P
This study presents physical and kinetic evidence for the interaction of GTR and GSAT and substrate channeling of the intermediate glutamate 1-semialdehyde (GSA) in the formation of ALA from glutamyltRNA

Summary

EXPERIMENTAL PROCEDURES

C. reinhardtii Enzymes and Antibodies to the Enzymes—A full-length cDNA clone encoding GSAT was previously isolated and cloned into pBSIISKϩ plasmid [14]. Expression of recombinant GSAT containing N-terminal His tag was induced with 1 mM isopropyl ␤-Dthiogalactopyranoside for 2 h at 37 °C. The product was purified under native conditions using Ni-NTA-agarose (Qiagen). A catalytically active expression construct of GTR corresponding to the open reading frame encoded by exon 2 of the GTR gene and containing a C-terminal His tag was previously described [15]. Expression of this construct was induced with 0.2 mM isopropyl-1-thio-␤-D-galactopyranoside for 15 h at 26 °C. The product was purified under native conditions using Ni-NTA-agarose and used for the generation of poly-

Protein Interactions in ALA Formation

RESULTS

Another possible alternative explanation for the inhibitory

DISCUSSION