Polyamines are important regulators in all living organisms and are implicated in essential biological processes including cell growth, differentiation and apoptosis. Pseudomonas aeruginosa possesses an spuABCDEFGHI gene cluster that is involved in the metabolism and uptake of two polyamines: spermidine and putrescine. In the proposed γ-glutamylation–putrescine metabolism pathway, SpuA hydrolyzes γ-glutamyl-γ-aminobutyrate (γ-Glu-GABA) to glutamate and γ-aminobutyric acid (GABA). In this study, crystal structures of P. aeruginosa SpuA are reported, confirming it to be a member of the class I glutamine amidotransferase (GAT) family. Activity and substrate-binding assays confirm that SpuA exhibits a preference for γ-Glu-GABA as a substrate. Structures of an inactive H221N mutant were determined with bound glutamate thioester intermediate or glutamate product, thus delineating the active site and substrate-binding pocket and elucidating the catalytic mechanism. The crystal structure of another bacterial member of the class I GAT family from Mycolicibacterium smegmatis (MsGATase) in complex with glutamine was determined for comparison and reveals a binding site for glutamine. Activity assays confirm that MsGATase has activity for glutamine as a substrate but not for γ-Glu-GABA. The work reported here provides a starting point for further investigation of polyamine metabolism in P. aeruginosa.

中文翻译：

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铜绿假单胞菌γ-谷氨酰-γ-氨基丁酸水解酶SpuA的结构与机制

多胺是所有活生物体中的重要调节剂，并且与包括细胞生长、分化和细胞凋亡在内的基本生物过程有关。铜绿假单胞菌拥有一个spuABCDEFGHI基因簇，该基因簇参与两种多胺的代谢和摄取：亚精胺和腐胺。在提出的 γ-谷氨酰化-腐胺代谢途径中，SpuA 将 γ-谷氨酰-γ-氨基丁酸 (γ-Glu-GABA) 水解为谷氨酸和 γ-氨基丁酸 (GABA)。在这项研究中，铜绿假单胞菌的晶体结构报道了 SpuA，证实它是 I 类谷氨酰胺氨基转移酶 (GAT) 家族的成员。活性和底物结合测定证实 SpuA 表现出对 γ-Glu-GABA 作为底物的偏好。用结合的谷氨酸硫酯中间体或谷氨酸产物确定无活性 H221N 突变体的结构，从而描绘活性位点和底物结合口袋并阐明催化机制。耻垢分枝杆菌I 类 GAT 家族的另一个细菌成员的晶体结构(MsGATase) 与谷氨酰胺复合物被确定用于比较，并揭示了谷氨酰胺的结合位点。活性测定证实 MsGATase 对作为底物的谷氨酰胺具有活性，但对 γ-Glu-GABA 没有活性。这里报道的工作为进一步研究铜绿假单胞菌中的多胺代谢提供了一个起点。