Identification and characterization of a heat-labile type I glutamine synthetase from Streptomyces cinnamonensis

Streptomycetes have two distinct glutamine synthetases (GS): a heat-stable dodecameric GSI and a heat-labile octameric GSII. A heat-inactivated GS activity was detected in crude extracts of Streptomyces cinnamonensis cells grown with nitrate or glutamate as the nitrogen source. The purified enzyme obtained from crude extracts of the nitrate-grown cells after affinity and anion-exchange chromatography was also heat-labile; it was inactivated by 80% when incubated at 50 degrees C for 1h. However, the enzyme has properties typical of GSI and similar with those of the heat-stable GSI purified from S. aureofaciens: It is composed of twelve subunits, each of M55 kDa, and has a native molar mass of 625 kDa and an isoelectric point at pH 4.2. In addition, its activity is regulated by reversible adenylylation. Mg2+ and NaCl but not Mn2+ protected the purified enzyme from thermal inactivation, and both NaCl and Mn2+ or Mg2+ stabilized its activity at 4-8 degrees C. As compared with GSI from S. aureofaciens, the S. cinnamonensis enzyme was cleaved more extensively during SDS-PAGE, was less sensitive to feedback inhibitors, and similarly affected by divalent cations. The K-m values were 125 mmol/L for L-glutamate, 0.1 for NH4+, 1.25 for ATP, 18.5 for L-glutamine, 3.3 for hydroxylamine and 0.087 for ADP. To our best knowledge, this is the first report of a heat-labile GSI from any source.