title: Escherichia coli glutamyl-tRNA reductase. Trapping the thioester intermediate.
abstract: In the first step of tetrapyrrole biosynthesis in Escherichia coli, glutamyl-tRNA reductase (GluTR, encoded by hemA) catalyzes the NADPH-dependent reduction of glutamyl-tRNA to glutamate-1-semialdehyde. Soluble homodimeric E. coli GluTR was made by co-expressing the hemA gene and the chaperone genes dnaJK and grpE. During Mg(2+)-stimulated catalysis, the reactive sulfhydryl group of Cys-50 in the E. coli enzyme attacks the alpha-carbonyl group of the tRNA-bound glutamate. The resulting thioester intermediate was trapped and detected by autoradiography. In the presence of NADPH, the end product, glutamate-1-semialdehyde, is formed. In the absence of NADPH, E. coli GluTR exhibited substrate esterase activity. The in vitro synthesized unmodified glutamyl-tRNA was an acceptable substrate for E. coli GluTR. Eight 5-aminolevulinic acid auxotrophic E. coli hemA mutants were genetically selected, and the corresponding mutations were determined. Most of the recombinant purified mutant GluTR enzymes lacked detectable activity. Based on the Methanopyrus kandleri GluTR structure, the positions of the amino acid exchanges are close to the catalytic domain (G7D, E114K, R314C, S22L/S164F, G44C/S105N/A326T, G106N, S145F). Only GluTR G191D (affected in NADPH binding) revealed esterase but no reductase activity.
authors.affiliation: Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schauer
authors.forename: Shalini
authors.initials: S
authors.lastname: Chaturvedi
authors.forename: Lennart
authors.initials: L
authors.lastname: Randau
authors.forename: Jürgen
authors.initials: J
authors.lastname: Moser
authors.forename: Makoto
authors.initials: M
authors.lastname: Kitabatake
authors.forename: Stefan
authors.initials: S
authors.lastname: Lorenz
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Verkamp
authors.forename: Wolf-Dieter
authors.initials: WD
authors.lastname: Schubert
authors.forename: Toru
authors.initials: T
authors.lastname: Nakayashiki
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Murai
authors.forename: Kristen
authors.initials: K
authors.lastname: Wall
authors.forename: Hans-Ulrich
authors.initials: HU
authors.lastname: Thomann
authors.forename: Dirk W
authors.initials: DW
authors.lastname: Heinz
authors.forename: Hachiro
authors.initials: H
authors.lastname: Inokuchi
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
authors.forename: Dieter
authors.initials: D
authors.lastname: Jahn
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D004952:Esters; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D004952:Esters; D011994:Recombinant Proteins; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase