Novel inhibitors of glutamyl-tRNA(Glu) reductase identified through cell-based screening of the heme/chlorophyll biosynthetic pathway

• Published in: Archives of biochemistry and biophysics Vol. 372; no. 2; pp. 230 - 237
• Main Authors: Loida, P.J, Thompson, R.L, Walker, D.M, CaJacob, C.A
• Format: Journal Article
• Language: English
• Published: United States 15.12.1999
• Subjects: Aldehyde Oxidoreductases - antagonists & inhibitors; Aldehyde Oxidoreductases - metabolism; aminolevulinic acid; Aminolevulinic Acid - metabolism; Aminolevulinic Acid - pharmacology; Arabidopsis - genetics; biochemical pathways; chlorophyll; Chlorophyll - biosynthesis; Cyclohexanecarboxylic Acids - pharmacology; Drug Evaluation, Preclinical; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - classification; Enzyme Inhibitors - pharmacology; Escherichia coli - drug effects; Escherichia coli - genetics; Escherichia coli - growth & development; Escherichia coli - metabolism; Glutamates - metabolism; glutamic acid; heme; Heme - biosynthesis; Hordeum - enzymology; Hordeum - genetics; Inhibitory Concentration 50; Intramolecular Transferases - antagonists & inhibitors; Intramolecular Transferases - metabolism; Kinetics; Mutation - genetics; Recombinant Fusion Proteins - antagonists & inhibitors; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Reproducibility of Results; RNA, Transfer, Gln - metabolism; Sequence Homology, Amino Acid; Spinacia oleracea - genetics; Substrate Specificity; transfer RNA
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1999.1505

The metabolite 5-aminolevulinic acid (ALA) is an early committed intermediate in the biosynthetic pathway of heme and chlorophyll formation. In plants, 5-aminolevulinic acid is synthesized via a two-step pathway in which glutamyl-tRNA(Glu) is reduced by glutamyl-tRNA(Glu) reductase (GluTR) to glutamate 1-semialdehyde, followed by transformation to 5-aminolevulinic acid catalyzed by glutamate 1-semialdehyde aminotransferase. Using an Escherichia coli cell-based high-throughput assay to screen small molecule libraries, we identified several chemical classes that specifically inhibit heme/chlorophyll biosynthesis at this point by demonstrating that the observed cell growth inhibition is reversed by supplementing the medium with 5-aminolevulinic acid. These compounds were further tested in vitro for inhibition of the purified enzymes GluTR and glutamate 1-semialdehyde aminotransferase as confirmation of the specificity and site of action. Several promising compounds were identified from the high-throughput screen that inhibit GluTR with an I(0.5) of less than 10 microM. Our results demonstrate the efficacy of cell-based high-throughput screening for identifying inhibitors of 5-aminolevulinic acid biosynthesis, thus representing the first report of exogenous inhibitors of this enzyme.