IDENTIFICATION AND CHARACTERIZATION OF A HEAT-LABILE TYPE-I GLUTAMINE-SYNTHETASE FROM STREPTOMYCES-CINNAMONENSIS

Streptomycetes have two distinct glutamine synthetases (GS): a heat-st able dodecameric GSI and a heat-labile octameric GSII. A heat-inactiva ted GS activity was detected in crude extracts of Streptomyces cinnamo nensis cells grown with nitrate or glutamate as the nitrogen source. T he purified enzyme obtained from crude extracts of the nitrate-grown c ells after affinity and anion-exchange chromatography was also heat-la bile; it was inactivated by 80% when incubated at 50 degrees C for 1h. However, the enzyme has properties typical of GSI and similar with th ose of the heat-stable GSI purified from S. aureofaciens: It is compos ed 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 Mn2protected the purified enzyme from thermal inactivation, and both NaCl and Mn2+ or Mg2+ stabilized its activity at 4-8 degrees C. As compare d with GSI from S. aureofaciens, the S. cinnamonensis enzyme was cleav ed more extensively during SDS-PAGE, was less sensitive to feedback in hibitors, and similarly affected by divalent cations. The K-m values w ere 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 knowl edge, this is the first report of a heat-labile GSI from any source.