The hemA gene encoding glutamyl-tRNA reductase from the archaeon Methanobacterium thermoautotrophicum strain Marburg

Abstract

In archaea the first general tetrapyrrole precursor 5-aminolevulinic acid (ALA) is formed via the tRNA-dependent five-carbon pathway from glutamate. We have cloned the hemA gene encoding the central enzyme of the pathway glutamyl-tRNA reductase from the methanogenic archaeon Methanobacterium thermoautotrophicum by complementation of an Escherichia coli hemA mutant to ALA prototrophy. An 1194 bp open reading frame that encodes a 398 amino acid polypeptide with the calculated M, 44,509 was detected. The deduced amino acid sequence showed 20-35% amino acid identity to bacterial HemAs with the highest identity score to the Pseudomonas aeruginosa HemA. An identity of approximately 22% was found to plant HemAs. Glutamyl-tRNA reductase activity was shown for the M. thermoautotrophicum HemA after overexpression in E. coli and partial purification. The enzymatic reaction catalyzed by the partially purified enzyme revealed a temperature optimum of 65 degrees C at an optimal pH of 7.0. The reductase utilized preferentially NADPH for the reduction of the activated carboxyl group. The presence of ATP and GTP showed no obvious influence on catalysis.