Universidade Federal do Paraná - UFPR, Departamento de Bioquímica e Biologia Molecular, Núcleo de Fixação Biológica de Nitrogênio, Curitiba, PR, Brasil.
Universidade Federal do Paraná - UFPR, Setor Litoral, Caiobá Matinhos, PR, Brasil.
Braz J Biol. 2021 May 28;82:e235927. doi: 10.1590/1519-6984.235927. eCollection 2021.
Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.
谷氨酰胺合成酶（GS）由 glnA 编码，催化 L-谷氨酸和铵转化为 L-谷氨酰胺。这个由 ATP 水解驱动的过程是固氮菌 Azospirillum brasilense 中主要的氮同化途径。A. brasilense 菌株 HM053 的 GS 活性较差，在固氮条件下会将铵漏入培养基中。在这项工作中，野生型和 HM053 菌株的 glnA 基因被克隆到 pET28a 中，在大肠杆菌中进行了测序和过表达。GS 酶通过亲和层析进行纯化和表征。HM053 菌株的 GS 携带 P347L 取代，导致酶活性降低，并使酶对 GlnE 的腺苷酰化作用不敏感。
