Purification and partial characterisation of barley glutamyl-tRNA(Glu) reductase, the enzyme that directs glutamate to chlorophyll biosynthesis.
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 microM concentration or glutamate 1-semialdehyde at 200 microM caused a 50% inhibition of the reductase activity. Micromolar concentrations of Zn2+, Cu2+ and Cd2+ also inhibited barley glutamyl-tRNA(Glu) reductase.
Carlsberg Laboratory, Department of Physiology, Copenhagen-Valby, Denmark.
B
B
Pontoppidan
C G
CG
Kannangara
D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry; D002734:Chlorophyll / Q000096:biosynthesis*; D002736:Chloroplasts / Q000201:enzymology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D018698:Glutamic Acid / Q000096:biosynthesis*; D001467:Hordeum / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid
D002734:Chlorophyll; D018698:Glutamic Acid; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase