Complex Formation between Glutamyl-tRNA Reductase and Glutamate-1-semialdehyde 2,1-Aminomutase in Escherichia coli during the Initial Reactions of Porphyrin Biosynthesis

Abstract

In Escherichia coli the first common precursor of all tetrapyrroles, 5-aminolevulinic acid, is synthesized from glutamyl-tRNA (Glu-tRNA Glu ) in a two-step reaction catalyzed by glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde 2,1-aminomutase (GSA-AM). To protect the highly reactive reaction intermediate glutamate-1-semialdehyde (GSA), a tight complex between these two enzymes was proposed based on their solved crystal structures. The existence of this hypothetical complex was verified by two independent biochemical techniques. Co-immunoprecipitation experiments using antibodies directed against E. coli GluTR and GSA-AM demonstrated the physical interaction of both enzymes in E. coli cell-free extracts and between the recombinant purified enzymes. Additionally, the formation of a GluTR⅐GSA-AM complex was identified by gel permeation chromatography. Complex formation was found independent of Glu-tRNA Glu and cofactors. The analysis of a GluTR mutant truncated in the 80-amino acid C-terminal dimerization domain (GluTR-A338Stop) revealed the importance of GluTR dimerization for complex formation. The in silico model of the E. coli GluTR⅐GSA-AM complex suggested direct metabolic channeling between both enzymes to protect the reactive aldehyde species GSA. In accordance with this proposal, side product formation catalyzed by GluTR was observed via high performance liquid chromatography analysis in the absence of the GluTR⅐GSA-AM complex.In plants, green algae, archaea, and most bacteria the common precursor molecule of all tetrapyrroles, 5-aminolevulinic acid (ALA), 1 is synthesized from tRNA-bound glutamate (GlutRNA Glu ) in a two-step reaction (1, 2). In the first step, the NADPH-dependent glutamyl-tRNA reductase (GluTR) catalyzes the reduction of glutamyl-tRNA to glutamate-1-semialdehyde (GSA). In a subsequent transamination reaction this aldehyde is transformed into ALA by the pyridoxal 5Ј-phosphatedependent enzyme glutamate-1-semialdehyde 2,1-aminomutase (GSA-AM) (3).Recently the catalytic mechanism of GluTR and its structural basis have been elucidated in a combined biochemical and structural investigation using the recombinant enzyme from the extreme thermophilic archaean Methanopyrus kandleri (4, 5). The crystal structure of GluTR reveals an unusual extended V-shaped dimer with each monomer consisting of three distinct domains arranged along a curved "spinal" ␣-helix. The N-terminal catalytic domain specifically recognizes the glutamate moiety of the substrate. The active site was identified by cocrystallization of the competitive inhibitor glutamycin representing the 3Ј-terminal end of the natural substrate. During catalysis a nucleophilic cysteine residue attacks the aminoacyl linkage of the glutamate to its cognate tRNA and generates an enzyme-bound thioester intermediate. This intermediate has been biochemically trapped and visualized. For this purpose a new purification strategy for the Escherichia coli enzyme has been developed (6, 7). The thioester intermediate gets finally reduced by direc...

Crystal structure of Arabidopsis glutamyl-tRNA reductase in complex with its stimulator protein

“…1A and 1B and Table S1). Surprisingly, GluBP binds directly to the cleft of the V-shaped GluTR dimer, in a manner similar to that which has been proposed for the GluTR-GSAM complex (11,15,16,18,19). This is contrary to the assumption that GluBP does not interfere with the GluTR-GSAM interaction, and therefore GluBP attaches to GluTR's NADPH-binding domain (10) or dimerization domain (20).…”

“…This glycine is also strictly conserved, suggesting that the tunnel observed here may be a common feature for GluTR. The tunnel observed for GSA release in the GluTR-GluBP complex verifies the proposed "back door" for GSA channeling to GSAM, thus providing evidence for a theoretical model developed earlier (11,(14)(15)(16)18). GluBP Stimulates GluTR Activity and Regulates GSA Release.…”

Tetrapyrrole Metabolism inArabidopsis thaliana

“…From the determined structure of Synechocystis GSA-AT (Hennig et al 1997), this enzyme is proposed to form a complex with GluTR, which may prevent the release of the highly-reactive aldehyde moiety of GSA by direct channeling of this intermediate from GluTR to GSA-AT (Moser et al 2001). Physical and kinetic interactions between GluTR and GSA-AT have been demonstrated using recombinant proteins of Chlamydomonas and E. coli (Luer et al 2005).…”

N-terminal engineering of glutamyl-tRNA reductase with positive charge arginine to increase 5-aminolevulinic acid biosynthesis

“…1). 28 Consequently, in order to further improve the stability of the enzyme, different numbers of lysine were introduced at the N-terminus of GluTR, respectively ( Fig. 2A).…”