Purification and partial characterisation of barley glutamyl‐tRNAGlu reductase, the enzyme that directs glutamate to chlorophyll biosynthesis

Abstract

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‐tRNAGlu to glutamate 1‐semialdehyde and tRNAGlu. 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‐tRNAGlu 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‐tRNAGlu 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 Zn2+, Cu2+ and Cd2+ also inhibited barley glutamyl‐tRNAGlu reductase. Copyright © 1994, Wiley Blackwell. All rights reserved