Open AccessPurification and partial characterisation of barley glutamyl‐tRNA Glu reductase, the enzyme that directs glutamate to chlorophyll biosynthesis
Author(s) -
Pontoppidan Bo,
Kannangara C. Gamini
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.00529.x
Subject(s) - biochemistry , reductase , biology , transfer rna , enzyme , complementary dna , amino acid , biosynthesis , hordeum vulgare , chloroplast , protochlorophyllide , rna , gene , botany , poaceae
5‐Aminolevulinic acid for chlorophyll synthesis in greening barley is formed from glutamate. One of the steps involved in the conversion of glutamate to 5‐aminolevulinic acid involves a reduction of glutamyl‐tRNA Glu to glutamate 1‐semialdehyde and tRNA Glu . An enzyme catalysing this reduction was purified from the stroma of greening barley chloroplasts. An approximately 270‐kDa protein composed of 54‐kDa identical subunits was identified as the barley glutamyl‐tRNA Glu reductase after purification by Sephacryl S‐300, Cibacron Blue‐Sepharose, 2′‐5′‐ADP‐Sepharose, Mono S, Mini Q and Superose 12 chromatography. The sequence of 18 amino acids from the N‐terminus of the reductase is 50% identical to a cDNA‐deduced domain of the Arabidopsis thaliana hemA protein and encoded in a barley hemA cDNA sequence. This is an unequivocal demonstration that the glutamyl‐tRNA Glu reductase subunit of higher plants is encoded in a hemA gene of the nuclear genome. Heme at 4 μM concentration or glutamate 1‐semialdehyde at 200 μM caused a 50% inhibition of the reductase activity. Micromolar concentrations of Zn 2+ , Cu 2+ and Cd 2+ also inhibited barley glutamyl‐tRNA Glu reductase.