对铜绿假单胞菌酰胺酶预测的半胱氨酸-谷氨酸-赖氨酸催化三联体三维结构的支持来自定点诱变和改变底物特异性的突变。
Support for a three-dimensional structure predicting a Cys-Glu-Lys catalytic triad for Pseudomonas aeruginosa amidase comes from site-directed mutagenesis and mutations altering substrate specificity.
作者信息
Novo Carlos, Farnaud Sebastien, Tata Renée, Clemente Alda, Brown Paul R
机构信息
Unidade de Tecnologica de Proteínas e Anticorpos Monoclonais, Departmento de Biotecnologia, Instituto Nacional de Engenharia e Tecnologia Industrial, Edifício F, Estrada do Paço do Lumiar 1649-038, Lisbon, Portugal.
出版信息
Biochem J. 2002 Aug 1;365(Pt 3):731-8. doi: 10.1042/BJ20011714.
Abstract
The aliphatic amidase from Pseudomonas aeruginosa belongs to the nitrilase superfamily, and Cys(166) is the nucleophile of the catalytic mechanism. A model of amidase was built by comparative modelling using the crystal structure of the worm nitrilase-fragile histidine triad fusion protein (NitFhit; Protein Data Bank accession number 1EMS) as a template. The amidase model predicted a catalytic triad (Cys-Glu-Lys) situated at the bottom of a pocket and identical with the presumptive catalytic triad of NitFhit. Three-dimensional models for other amidases belonging to the nitrilase superfamily also predicted Cys-Glu-Lys catalytic triads. Support for the structure for the P. aeruginosa amidase came from site-direct mutagenesis and from the locations of amino acid residues that altered substrate specificity or binding when mutated.
摘要
铜绿假单胞菌的脂肪族酰胺酶属于腈水解酶超家族，半胱氨酸（166）是催化机制中的亲核试剂。以线虫腈水解酶-脆性组氨酸三联体融合蛋白（NitFhit；蛋白质数据库登录号1EMS）的晶体结构为模板，通过比较建模构建了酰胺酶模型。酰胺酶模型预测了位于口袋底部的催化三联体（半胱氨酸-谷氨酸-赖氨酸），与NitFhit的推测催化三联体相同。属于腈水解酶超家族的其他酰胺酶的三维模型也预测了半胱氨酸-谷氨酸-赖氨酸催化三联体。对铜绿假单胞菌酰胺酶结构的支持来自定点诱变以及突变时改变底物特异性或结合的氨基酸残基的位置。
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