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Benzoic Acid 2-Hydroxylase, a Soluble Oxygenase from Tobacco, Catalyzes Salicylic Acid Biosynthesis.

Proceedings of the National Academy of Sciences of the United States of America(1995)SCI 1区

RUTGERS STATE UNIV

Cited 245|Views13
Abstract
Benzoic acid 2-hydroxylase (BA2H) catalyzes the biosynthesis of salicylic acid from benzoic acid. The enzyme has been partially purified and characterized as a soluble protein of 160 kDa. High-efficiency in vivo labeling of salicylic acid with 18O2 suggested that BA2H is an oxygenase that specifically hydroxylates the ortho position of benzoic acid. The enzyme was strongly induced by either tobacco mosaic virus inoculation or benzoic acid infiltration of tobacco leaves and it was inhibited by CO and other inhibitors of cytochrome P450 hydroxylases. The BA2H activity was immunodepleted by antibodies raised against SU2, a soluble cytochrome P450 from Streptomyces griseolus. The anti-SU2 antibodies immunoprecipitated a radiolabeled polypeptide of around 160 kDa from the soluble protein extracts of L-[35S]-methionine-fed tobacco leaves. Purified BA2H showed CO-difference spectra with a maximum at 457 nm. These data suggest that BA2H belongs to a novel class of soluble, high molecular weight cytochrome P450 enzymes.
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NICOTIANA TABACUM,TOBACCO MOSAIC VIRUS,CYTOCHROME P450,ACQUIRED RESISTANCE
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要点】:本文研究了从烟草中提取的苯甲酸2-羟基酶(BA2H)催化苯甲酸生成水杨酸的生物合成过程,揭示了BA2H作为一种新型可溶性高分子量细胞色素P450酶类的特性。

方法】:通过部分纯化和表征BA2H作为160 kDa的可溶性蛋白,并使用高效体内标记技术来确定其为一种专门羟基化苯甲酸邻位的氧合酶。

实验】:通过烟草叶片接种烟草花叶病毒或用苯甲酸浸润处理强烈诱导BA2H酶的生成,并通过CO和其他细胞色素P450羟基酶抑制剂来测试其活性;使用针对SU2(来自灰色链霉菌的可溶性细胞色素P450)的抗体进行免疫耗竭实验,发现BA2H的活性可以被这些抗体降低,且能从用L-[35S]-蛋氨酸喂养的烟草叶片可溶性蛋白提取物中免疫沉淀出约160 kDa的放射性标记多肽;纯化的BA2H在457 nm处显示CO差异光谱。实验使用的数据集未在文本中明确提及。