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 archacon 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 r 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 °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. The cloning and characterization of the hemA gene from Methanobacterium thermoautotrophicum encoding glutamyl tRNA reductase is reported. An initial biochemical characterization of the recombinant HemA protein is described.
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