Control of substrate specificity by active-site residues in nitrobenzene dioxygenase
Control of substrate specificity by active-site residues in nitrobenzene dioxygenase
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DOI：
10.1128/aem.72.3.1817-1824.2006
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发表时间：
2006-03-01
影响因子：
4.4
通讯作者：
Parales, RE
中科院分区：
文献类型：
--
作者：
Ju, KS;Parales, RE
来自丛毛单胞菌JS 765的硝基苯1，2-双加氧酶催化硝基苯降解的初始反应，生成邻苯二酚和亚硝酸盐。该酶还氧化硝基取代碳上的单硝基甲苯和二硝基甲苯的芳环，但这种特异性的基础尚不清楚。本研究采用定点突变的方法对硝基苯双加氧酶的活性位点进行修饰，并评价了特定残基在控制底物特异性和酶性能方面的贡献。六个突变体酶的活动表明，在位置258，293，和350的α亚基的残基是重要的，以确定与硝基芳烃底物的区域特异性和对映体特异性与萘。这些结果为硝基芳烃底物的特征特异性提供了解释。基于硝基苯双加氧酶的结构，在258位用缬氨酸取代天冬酰胺应消除底物硝基和天冬酰胺的氨基之间的氢键。由N258 V突变体形成的高达99%的一硝基甲苯氧化产物是硝基苄醇而不是儿茶酚，支持这种氢键在定位环氧化活性位点中的底物中的重要性。I350 F突变体也得到了类似的结果，其中氢键的形成似乎被空间干扰所阻止。在位置293处具有取代的酶的特异性根据存在的残基而变化。与野生型相比，F293 Q突变体在氧化2，6-二硝基甲苯时快2.5倍，同时基于产物形成速率和全细胞动力学保留了类似的底物Km。
Nitrobenzene 1,2-dioxygenase from Comamonas sp. strain JS765 catalyzes the initial reaction in nitrobenzene degradation, forming catechol and nitrite. The enzyme also oxidizes the aromatic rings of mono- and dinitrotoluenes at the nitro-substituted carbon, but the basis for this specificity is not understood. In this study, site-directed mutagenesis was used to modify the active site of nitrobenzene dioxygenase, and the contribution of specific residues in controlling substrate specificity and enzyme performance was evaluated. The activities of six mutant enzymes indicated that the residues at positions 258, 293, and 350 in the alpha subunit are important for determining regiospecificity with nitroarene substrates and enantiospecificity with naphthalene. The results provide an explanation for the characteristic specificity with nitroarene substrates. Based on the structure of nitrobenzene dioxygenase, substitution of valine for the asparagine at position 258 should eliminate a hydrogen bond between the substrate nitro group and the amino group of asparagine. Up to 99% of the mononitrotoluene oxidation products formed by the N258V mutant were nitrobenzyl alcohols rather than catechols, supporting the importance of this hydrogen bond in positioning substrates in the active site for ring oxidation. Similar results were obtained with an I350F mutant, where the formation of the hydrogen bond appeared to be prevented by steric interference. The specificity of enzymes with substitutions at position 293 varied depending on the residue present. Compared to the wild type, the F293Q mutant was 2.5 times faster at oxidizing 2,6-dinitrotoluene while retaining a similar K-m for the substrate based on product formation rates and whole-cell kinetics.