Structure and mechanism of the gamma-glutamyl-gamma-aminobutyrate hydrolase SpuA from Pseuaomonas aeruginosa
Structure and mechanism of the gamma-glutamyl-gamma-aminobutyrate hydrolase SpuA from Pseuaomonas aeruginosa
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铜绿假单胞菌γ-谷氨酰-γ-氨基丁酸水解酶SpuA的结构和机制
DOI：
10.1107/s2059798321008986
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发表时间：
2021
影响因子：
2.2
通讯作者：
Bartlam Mark
中科院分区：
文献类型：
--
作者：
Chen Yujing;Jia Haizhu;Zhang Jianyu;Liang Yakun;Liu Ruihua;Zhang Qionglin;Bartlam Mark
多胺是所有生物体内重要的调节因子，参与细胞生长、分化和凋亡等重要生物过程。铜绿假单胞菌具有一个参与亚精胺和腐胺两种多胺代谢和摄取的spuABCDEFGHI基因簇。在提出的γ-谷氨酰基-腐胺代谢途径中，SpuA水解γ-谷氨酰基-γ-氨基丁酸（γ-Glu-GABA）生成谷氨酸和γ-氨基丁酸（GABA）。本研究报道了P. aeruginosa SpuA的晶体结构，证实其为I类谷氨酰胺氨基转移酶（GAT）家族成员。活性和底物结合实验证实SpuA表现出对γ-Glu-GABA作为底物的偏好。用结合谷氨酸硫酯中间体或谷氨酸产物测定失活H221N突变体的结构，从而描绘了活性位点和底物结合袋，并阐明了催化机制。对耻垢分枝杆菌（MsGATase）中另一种GAT家族I类细菌（MsGATase）与谷氨酰胺复合物的晶体结构进行了比较，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有活性，但对γ- γ-氨基丁酸没有活性。本研究为铜绿假单胞菌多胺代谢的进一步研究提供了一个起点。
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.