title: 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 GluTR.GSA-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 GluTR.GSA-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 GluTR.GSA-AM complex.
authors.affiliation: Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany.
authors.forename: Corinna
authors.initials: C
authors.lastname: Lüer
authors.forename: Stefan
authors.initials: S
authors.lastname: Schauer
authors.forename: Kalle
authors.initials: K
authors.lastname: Möbius
authors.forename: Jörg
authors.initials: J
authors.lastname: Schulze
authors.forename: Wolf-Dieter
authors.initials: WD
authors.lastname: Schubert
authors.forename: Dirk W
authors.initials: DW
authors.lastname: Heinz
authors.forename: Dieter
authors.initials: D
authors.lastname: Jahn
authors.forename: Jürgen
authors.initials: J
authors.lastname: Moser
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry*; D000447:Aldehydes / Q000737:chemistry; D015153:Blotting, Western; D002384:Catalysis; D002474:Cell-Free System; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D017353:Gene Deletion; D005971:Glutamates / Q000737:chemistry; D015151:Immunoblotting; D047468:Immunoprecipitation; D019751:Intramolecular Transferases / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011166:Porphyrins / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors
chemical_list: D000447:Aldehydes; D005971:Glutamates; D011166:Porphyrins; D011994:Recombinant Proteins; C057794:glutamate-1-semialdehyde; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase