BARLEY GLUTAMYL TRNA(GLU) REDUCTASE - MUTATIONS AFFECTING HEME INHIBITION AND ENZYME-ACTIVITY

Glutamyl tRNA(Glu) reductase converts glutamate molecules that are lig ated at their a-carboxyl groups to tRNA(Glu) into glutamate l-semialde hyde, an intermediate in the synthesis of 5-aminolevulinate, chlorophy ll and haem. The mature plant enzymes contain a highly conserved exten sion of 31-34 amino acids at the N-terminus not present in bacterial e nzymes. It is shown that barley glutamyl tRNA(Glu) reductases with a d eletion of the 30 N-terminal amino acids have the same high specific a ctivity as the untruncated enzymes, but are highly resistant to feed-b ack inhibition by haem. This peptide domain thus interacts directly or indirectly with haem and the toxicity of the 30 amino acid peptide fo r Escherichia coli experienced in mutant rescue and overexpression exp eriments can be explained by extensive haem removal from the metabolic pools that cannot be tolerated by the cell. Induced missense mutation s identify nine amino acids in the 451 residue long C-terminal part of the barley glutamyl tRNA(Glu) reductase which upon substitution curta il drastically, but do not eliminate entirely the catalytic activity o f the enzyme. These amino acids are thus important for the catalytic r eaction or tRNA binding. (C) 1997 Elsevier Science Ltd. All rights res erved.