Assembly of the Pseudomonas aeruginosa nonribosomal peptide siderophore pyochelin:: In vitro reconstitution of aryl-4,2-bisthiazoline synthetase activity from PchD, PPchE, and PchF
Assembly of the Pseudomonas aeruginosa nonribosomal peptide siderophore pyochelin:: In vitro reconstitution of aryl-4,2-bisthiazoline synthetase activity from PchD, PPchE, and PchF
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DOI：
10.1021/bi991787c
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发表时间：
1999-11-09
期刊：
影响因子：
2.9
通讯作者：
Walsh, CT
中科院分区：
文献类型：
--
作者：
Quadri, LEN;Keating, TA;Walsh, CT
参与非核糖体肽铁载体绿脓杆菌螯铁蛋白生物发生的三种铜绿假单胞菌蛋白质PchD、PchE和PchF已在大肠杆菌中表达并纯化，并发现其产生三环酸羟基苯基-噻唑基-噻唑啉基-羧酸（HPTT-COOH），这是一种含有铁载体的双噻唑啉类的芳基-4，2-双杂环骨架的高级中间体。这三种蛋白质含有三个腺苷酸化结构域，一个特异于水杨酸活化，两个特异于半胱氨酸活化，以及三个载体蛋白结构域（两个在PchE中，一个在PchF中），其经历翻译后引发，用磷酸泛酰巯基乙胺使水杨基和半胱氨酰部分能够共价连接作为酰基-S-酶中间体，两个环化结构域（PchE中的Cyl和PchF中的Cy 2）在延长链中产生两个酰胺键，并且酰基半胱氨酸部分环化脱水成噻唑啉环。第九个结构域是PchF中最下游的结构域，是链终止的酰基-S-酶硫酯水解酶，其将HPTT-S-酶中间体释放为观察到的串联双杂环酸产物。一个PchF-硫酯酶结构域活性位点双突变体未能翻转，但单环羟基苯基-噻唑啉基-半胱氨酸（HPT-Cys）的产品继续从PchE释放，允许分配的级联的酰基-S-酶的中间体参与的起始，延伸和终止步骤。
Three Pseudomonas aeruginosa proteins involved in biogenesis of the nonribosomal peptide siderophore pyochelin, PchD, PchE, and PchF, have been expressed in and purified from Escherichia coli and are found to produce the tricyclic acid hydroxyphenyl-thiazolyl-thiazolinyl-carboxylic acid (HPTT-COOH), an advanced intermediate containing the aryl-4,2-bis-heterocyclic skeleton of the bithiazoline class of siderophores. The three proteins contain three adenylation domains, one specific for salicylate activation and two specific for cysteine activation, and three carrier protein domains (two in PchE and one in PchF) that undergo posttranslational priming With phosphopantetheine to enable covalent-tethering of salicyl and cysteinyl moieties as acyl-S-enzyme intermediates,Two cyclization domains (Cyl in PchE and Cy2 in PchF) create the two amide linkages in the elongating chains and the cyclodehydrations of acylcysteine moieties into thiazolinyl rings. The ninth domain, the most downstream domain in PchF, is the chain-terminating, acyl-S-enzyme thioester hydrolase that releases the HPTT-S-enzyme intermediate to the observed tandem bis-heterocyclic acid product. A PchF-thioesterase domain active site double mutant fails to turn over, but a monocyclic hydroxyphenyl-thiazolinyl-cysteine (HPT-Cys) product continues to be released from PchE, allowing assignment of the cascade of acyl-S-enzyme intermediates involved in initiation, elongation, and termination steps.