谷氨酸棒状杆菌中龙胆酸1,2-双加氧酶底物范围的扩大,用于单羟基苯甲酸盐的转化

E. Eppinger, A. Stolz
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引用次数: 6

摘要

来自谷氨棒状杆菌和其他生物的龙胆酸1,2-双加氧酶(GDOs)氧化裂解龙胆酸(2,5-二羟基苯甲酸酯)的芳香核,但不能转化水杨酸(2-羟基苯甲酸酯)。相反,变形杆菌水杨酸假氨基杆菌合成一种酶(“水杨酸双加氧酶”,SDO),它可以裂解龙胆酸,但也可以(取代)水杨酸。序列比较表明,该GDO与C. glutamum ATCC 13032的GDO同属于主要来源于革兰氏阳性菌的GDO群。将序列比较与先前对SDO进行的结构和突变分析相结合,可以确定一个氨基酸残基(Ala112),该残基可能阻止谷氨酸丙氨酸GDO氧化(取代)水杨酸盐。因此,将相关突变(Ala→Gly)引入谷氨酸丙氨酸GDO中。获得的GDO变体获得了氧化水杨酸酯和其他几种单羟基化底物的能力。为了筛选更广泛的酶变异,一种显色试验被开发出来,允许检测转化水杨酸的细菌菌落。通过对不同位置饱和诱变获得的一组GDO变异进行筛选,验证了该检测系统的适用性。这表明携带突变Ala112→Ser、Ala112→Ile和Ala112→Asp的GDO变体也转化为水杨酸盐。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Expansion of the substrate range of the gentisate 1,2-dioxygenase from Corynebacterium glutamicum for the conversion of monohydroxylated benzoates
The gentisate 1,2-dioxygenases (GDOs) from Corynebacterium glutamicum and various other organisms oxidatively cleave the aromatic nucleus of gentisate (2,5-dihydroxybenzoate), but are not able to convert salicylate (2-hydroxybenzoate). In contrast, the &agr;-proteobacterium Pseudaminobacter salicylatoxidans synthesises an enzyme (‘salicylate dioxygenase’, SDO) which cleaves gentisate, but also (substituted) salicylate(s). Sequence comparisons showed that the SDO belongs to a group of GDOs mainly originating from Gram-positive bacteria which also include the GDO from C. glutamicum ATCC 13032. The combination of sequence comparisons with previously performed structural and mutational analyses of the SDO allowed to identify an amino acid residue (Ala112) which might prevent the oxidation of (substituted) salicylate(s) by the GDO from C. glutamicum. Therefore, the relevant mutation (Ala→Gly) was introduced into the GDO from C. glutamicum. The GDO variant obtained gained the ability to oxidise salicylate and several other monohydroxylated substrates. In order to screen a broader range of enzyme variants a chromogenic assay was developed which allowed the detection of bacterial colonies converting salicylate. The applicability of this test system was proven by screening a set of GDO variants obtained by saturation mutagenesis at different positions. This demonstrated that also GDO variants carrying the mutations Ala112→Ser, Ala112→Ile and Ala112→Asp converted salicylate.
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