吸水链霉菌井冈变种谷氨酰胺合成酶的研究

本文证明吸水链霉菌井冈变种产生一重要的农用抗生素——井冈霉素,生物合成中抗生素产量与菌体谷氨酰胺合成酶有正相关性,因此我们对这一酶进行了纯化,并研究了它的理化性质和调节特征。此酶分子量为530000,亚基分子量为43000;电子显微镜观察结果证明酶由十二个亚基组成,呈双层正六角形排列,酶活力最适pH为7.2,并需Mg~(2 )或Mn~(2 )作为辅因子。此酶受代谢产物的反馈抑制和积累反馈抑制;同时也受腺苷酰化和去腺苷酰化的共价键调节,即当酶处在腺苷酰化状态,保持低活性,去除腺苷酰基后可恢复其高活性,这一结果为谷氨酰胺合成酶共价键修饰存在于革兰氏阳性细菌提供了有力的例证。     

THE ROUTE OF INCORPORATION OF NITROGEN ATOM INTO RIFAMYCIN DURING ITS BIOSYNTHESIS

In this paper, it was first shown that under various conditions of nitrogen supply therifamycin yield was positively correlated with the mycelial specific activity of glutaminesynthetase (GS), then the enhancing effect of glutamine, the product of GS, on rifamycin bio-synthesis was demonstrated with resting cell system. The stimulatory effect of glutaminewas more pronounced than that of glutamate, and not reduced by a GS specific inhibitor, DON.However, the increase in yield brought about by glutamate, and by asparagine was stronglyinhibited by this inhibitor. Glutamine-CO~(15)NH_2 and glutamato-α-~(15)NH_2 were synthesized andcompared for the incorporation of ~(15)N into rifamycin. It was found that the amide nitrogenwas incorporated to a much greater extent than the α-amino nitrogen, showing that glutaminewas a direct precursor of the nitrogen atom in rifamycin. In addition, synthesis of A-32(C_7N), an intermediate secreted by an inactive mutant rif 1, was also greatly stimulatedby glutamine, and the synthetic C_7N was found to be able to stimulate the biosynthesis ofrifamycin. Based upon the above results, the route of incorporation of nitrogen atom into rifamycinis summarized as follows:     

STUDIES ON GLUTAMINE SYNTHETASE FROM Streptomyces hygroscopicus var. jinggangensis

Streptomyces hygroscopicus var. jinggangensis produces validamycin, an important antibiotic used in agriculture. It was found that thexe was a positive correlation between the specific activity of mycelial glutamine synthetase (GS) and validamycin biosynthesis in our laboratory. So, in this paper, the purification, characteristics and regulatory properties of GS are reported. The native enzyme had a molecular weight of approximately 530,000 and was composed of 12 identical subunits, each 43,000. Electronic microscopic examination of preparations negatively stained disclosed that the subunits were arranged in two hexagonal rings that lay one on top of the other in a face-to-face fashion. Mg~(2+) or Mn~(2+) was absolutely needed as cofactor for GS activity. The enzyme activity was regulated by feedback inhibition and cumulative feedback inhibition. In addition, it was also regulated through a covalent modification, adenylylation and deadenylylation, suggesting that the covalent modification of GS exists n     

力复霉素生物合成中氮原子的参入途径

本文报道力复霉素产量与谷氨酰胺合成酶(GS)的正相关性,进而在洗涤菌体实验中证明谷氨酰胺促进力复霉素合成的作用远比谷氨酸显著,而这一促进作用不受GS专一性抑制剂DON的抑制,其它氨基酸如谷氨酸门冬酰胺的促进作用则受到这一抑制剂的抑制。谷氨酰胺-CO~(15)NH_2与谷氨酸-α-~(15)NH_2参入到力复霉素的比较实验指出,前者是力复霉素更直接的前体。再者,A-32(C_7N)的合成受谷氨酰胺促进,而人工合成的A-32(C_7N)又能促进力复霉素的合成。根据以上结果,本文提出了力复霉素分子中氮原子参入的途径。