Biomaterial Database

Escherichia coli glutamyl-tRNA reductase. Trapping the thioester intermediate. 2002

Stefan Schauer, and Shalini Chaturvedi, and Lennart Randau, and Jürgen Moser, and Makoto Kitabatake, and Stefan Lorenz, and Elizabeth Verkamp, and Wolf-Dieter Schubert, and Toru Nakayashiki, and Masatoshi Murai, and Kristen Wall, and Hans-Ulrich Thomann, and Dirk W Heinz, and Hachiro Inokuchi, and Dieter Söll, and Dieter Jahn

Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, Germany.

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.

UI	MeSH Term	Description	Entries
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D008970	Molecular Weight	The sum of the weight of all the atoms in a molecule.	Molecular Weights,Weight, Molecular,Weights, Molecular
D011487	Protein Conformation	The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain).	Conformation, Protein,Conformations, Protein,Protein Conformations
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002384	Catalysis	The facilitation of a chemical reaction by material (catalyst) that is not consumed by the reaction.	Catalyses
D004591	Electrophoresis, Polyacrylamide Gel	Electrophoresis in which a polyacrylamide gel is used as the diffusion medium.	Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D004952	Esters	Compounds derived from organic or inorganic acids in which at least one hydroxyl group is replaced by an –O-alkyl or another organic group. They can be represented by the structure formula RCOOR’ and are usually formed by the reaction between an acid and an alcohol with elimination of water.	Ester
D000445	Aldehyde Oxidoreductases	Oxidoreductases that are specific for ALDEHYDES.	Aldehyde Oxidoreductase,Oxidoreductase, Aldehyde,Oxidoreductases, Aldehyde
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein