Escherichia coli glutamyl-tRNA reductase. Trapping the thioester intermediate.
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.
Schauer
Stefan
S
Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, Germany.
Chaturvedi
Shalini
S
Randau
Lennart
L
Moser
Jürgen
J
Kitabatake
Makoto
M
Lorenz
Stefan
S
Verkamp
Elizabeth
E
Schubert
Wolf-Dieter
WD
Nakayashiki
Toru
T
Murai
Masatoshi
M
Wall
Kristen
K
Thomann
Hans-Ulrich
HU
Heinz
Dirk W
DW
Inokuchi
Hachiro
H
Söll
Dieter
D
Jahn
Dieter
D
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
D017931:DNA Primers; D004952:Esters; D011994:Recombinant Proteins; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase