Purification and Properties of Glutamine Synthetase from Methylococcus capsulatus (Bath)

Abstract

The obligate methanotroph Methylococcus capsulatus (Bath) was grown in batch and continuous culture with ammonia, nitrate or dinitrogen as the sole nitrogen source. Cell-free extracts from these cultures all contained the ammonia assimilation enzyme glutamine synthetase. During growth on ammonia, however, the glutamine synthetase was biosynthetically inactive. Isolation of glutamine synthetase from M. capsulatus (Bath) resulted in a 133-fold purification with a yield of 11%. The purified enzyme had a molecular weight of 617000 and a subunit size of 60000 Dal. The metal ion and nucleotide specificity of glutamine synthetase was studied using both a biosynthetic assay and an artificial assay - the γ-glutamyltransferase assay. Feedback inhibition by several end-products of glutamine metabolism was observed and their effects were cumulative Control of glutamine synthetase activity by adenylylation/deadenylylation was cera demonstrated by snake venom phosphodiesterase treatment of the enzyme isolated from cells grown with different nitrogen sources.