A flavin-dependent tryptophan 6-halogenase and its use in modification of pyrrolnitrin biosynthesis
A flavin-dependent tryptophan 6-halogenase and its use in modification of pyrrolnitrin biosynthesis
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DOI：
10.1080/10242420601033738
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发表时间：
2006-11-01
影响因子：
1.8
通讯作者：
van Pee, Karl-Heinz
中科院分区：
文献类型：
--
作者：
Seibold, Corina;Schnerr, Helge;van Pee, Karl-Heinz
富电子底物的区域选择性卤化是由黄素依赖的卤化酶催化的。由白灰链霉菌产生的硫绿肽在吲哚环系统的6位含有一个氯原子，被认为是由色氨酸衍生而来。利用pyrrolnitrin生物合成基因簇中的色氨酸7-卤化酶（PrnA）基因克隆、测序并在假单胞菌中异源过表达色氨酸6-卤化酶基因。纯化酶的体外活性只能在由卤素酶、黄素还原酶、NADH、FAD和卤化物离子组成的双组分酶体系中表现出来。该酶催化l-和d-色氨酸的区域选择性氯化和溴化。在其天然形式的酶可能是一个同源二聚体，相对分子质量为63 600的亚基（包括his标签）。用含有色氨酸6-卤化酶基因的质粒转化硝基硝基假单胞菌ACN，生成新的氨基硝基硝基衍生物3-（2′-氨基-4′-氯苯基）吡咯。
Regioselective halogenation of electron rich substrates is catalysed by flavin-dependent halogenases. Thienodolin produced by Streptomyces albogriseolus contains a chlorine atom in the 6-position of the indole ring system and is believed to be derived from tryptophan. Using the gene of the tryptophan 7-halogenase (PrnA) from the pyrrolnitrin biosynthetic gene cluster the gene for a tryptophan 6-halogenase was cloned, sequenced and heterologously overexpressed in Pseudomonas strains. In vitro activity of the purified enzyme could only be shown in a two-component enzyme system consisting of the halogenase, a flavin reductase, NADH, FAD and halide ions. The enzyme catalyses the regioselective chlorination and bromination of l- and d-tryptophan. In its native form the enzyme is probably a homodimer with a relative molecular mass of the subunits of 63 600 (including the His-tag). Transformation of the pyrrolnitrin producer Pseudomonas chlororaphis ACN with a plasmid containing the tryptophan 6-halogenase gene lead to the formation of the new aminopyrrolnitrin derivative 3-(2'-amino-4'-chlorophenyl) pyrrole.