Hutcheson S W, Kosuge T
J Biol Chem. 1985 May 25;260(10):6281-7.
The oxidative decarboxylation of L-tryptophan to yield 3-indoleacetamide, catalyzed by tryptophan 2-monooxygenase, represents a controlling reaction in the synthesis of indoleacetic acid by Pseudomonas savastanoi (Pseudomonas syringae pv. savastanoi), a gall-forming pathogen of olive (Olea europea L.) and oleander (Nerium oleander L.). Production of indoleacetic acid is essential for virulence of the bacterium in its hosts. Tryptophan 2-monooxygenase was characterized to determine its role in indoleacetic acid metabolism in the bacterium. The enzyme was purified to apparent homogeneity from Escherichia coli cells containing the genetic locus for this enzyme obtained from P. savastanoi. The preparation contained a single polypeptide with a mass of 62,000 that cross-reacted immunologically with a homologous protein in P. savastanoi. The holoenzyme contained one FAD moiety/subunit with properties consistent with a catalytic function. The enzyme preparation catalyzed an L-tryptophan-dependent O2 uptake and yielded 3-indoleacetamide as a product. Enzyme activity fit simple Michaelis Menten kinetics with a Km for L-tryptophan of 50 microM. 3-Indoleacetamide and 3-indoleacetic acid were identified as regulatory effectors. The apparent Ki for 3-indoleacetamide was 7 microM; that for indoleacetic acid was 225 microM. At Km concentrations of tryptophan, enzyme activity was inhibited 50% by 25 microM 3-indoleacetamide. In contrast, 230 microM indoleacetic acid was required to effect a similar inhibition. Phenylalanine and tyrosine were ineffective as regulatory metabolites. These results indicate that IAA synthesis in P. savastanoi is regulated by limiting tryptophan and by feedback inhibition from indoleacetamide and indoleacetic acid.
色氨酸2-单加氧酶催化L-色氨酸氧化脱羧生成3-吲哚乙酰胺，这是丁香假单胞菌（丁香假单胞菌油橄榄致病变种）合成吲哚乙酸过程中的一个关键反应，该菌是橄榄（油橄榄）和夹竹桃（夹竹桃）上形成瘿瘤的病原体。吲哚乙酸的产生对于该细菌在宿主中的致病性至关重要。对色氨酸2-单加氧酶进行了表征，以确定其在细菌吲哚乙酸代谢中的作用。该酶从含有源自油橄榄假单胞菌的该酶基因位点的大肠杆菌细胞中纯化至表观均一。该制剂含有一种质量为62000的单一多肽，与油橄榄假单胞菌中的同源蛋白发生免疫交叉反应。全酶每个亚基含有一个FAD部分，其性质与催化功能一致。该酶制剂催化依赖L-色氨酸的氧气摄取，并产生3-吲哚乙酰胺作为产物。酶活性符合简单的米氏动力学，L-色氨酸的Km为50μM。3-吲哚乙酰胺和3-吲哚乙酸被鉴定为调节效应物。3-吲哚乙酰胺的表观Ki为7μM；吲哚乙酸的表观Ki为225μM。在色氨酸的Km浓度下，25μM的3-吲哚乙酰胺可抑制酶活性50%。相比之下，需要230μM的吲哚乙酸才能产生类似的抑制作用。苯丙氨酸和酪氨酸作为调节代谢物无效。这些结果表明，油橄榄假单胞菌中吲哚乙酸的合成受色氨酸限制以及吲哚乙酰胺和吲哚乙酸的反馈抑制调节。
