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首页> 外文期刊>Journal of biological inorganic chemistry: JBIC: a publication of the Society of Biological Inorganic Chemistry >Selective tuning of activity in a multifunctional enzyme as revealed in the F21W mutant of dehaloperoxidase B from Amphitrite ornata
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Selective tuning of activity in a multifunctional enzyme as revealed in the F21W mutant of dehaloperoxidase B from Amphitrite ornata

机译：从amphitrite ornata的除山氧化酶B的F21w突变体中揭示了多官能酶的活性调整

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摘要

Possessing both peroxidase and peroxygenase activities with a broad substrate profile that includes phenols, indoles, and pyrroles, the enzyme dehaloperoxidase (DHP) from Amphitrite ornata is a multifunctional catalytic hemoglobin that challenges many of the assumptions behind the well-established structure-function paradigm in hemoproteins. While previous studies have demonstrated that the F21W variant leads to attenuated peroxidase activity in DHP, here we have studied the impact of this mutation on peroxygenase activity to determine if it is possible to selectively tune DHP to favor one function over another. Biochemical assays with DHP B (F21W) revealed minimal decreases in peroxygenase activity of 1.2-2.1-fold as measured by 4-nitrophenol or 5-Br-indole substrate conversion, whereas the peroxidase activity catalytic efficiency for 2,4,6-trichlorophenol (TCP) was more than sevenfold decreased. Binding studies showed a 20-fold weaker affinity for 5-bromoindole (K (d) = 2960 +/- 940 mu M) in DHP B (F21W) compared to WT DHP B. Stopped-flow UV/visible studies and isotope labeling experiments together suggest that the F21W mutation neither significantly changes the nature of the catalytic intermediates, nor alters the mechanisms that have been established for peroxidase and peroxygenase activities in DHP. The X-ray crystal structure (1.96 ; PDB 5VLX) of DHP B (F21W) revealed that the tryptophan blocks one of the two identified TCP binding sites, specifically TCPinterior, suggesting that the other site, TCPexterior, remains viable for binding peroxygenase substrates. Taken together, these studies demonstrate that blocking the TCPinterior binding site in DHP selectively favors peroxygenase activity at the expense of its peroxidase activity.
机译：具有宽底物曲线的过氧化物酶和过氧化根酶活性，包括酚类，吲哚和胃溶解，来自amphitrite ornata的酶除卤氧化酶（DHP）是多功能催化血红蛋白，其挑战良好的结构功能范式背后的许多假设血红蛋白。虽然先前的研究表明，F21W变体导致DHP中的过氧化物酶活性导致衰减过氧化物酶活性，但在这里我们研究了该突变对过氧酶活性的影响，以确定是否可以选择性地调节DHP以支持一个功能。用DHP B（F21W）的生化测定显示通过4-硝基苯酚或5-BR-吲哚基材转化测量的过氧酶活性为1.2-2.1倍的最小降低，而2,4,6-三氯苯酚的过氧化物酶活性催化效率（ TCP）超过七倍降低。结合研究表明，与WT DHP B的DHP B（F21W）中的5-溴吲哚（K（D）= 2960 +/-940μm）显示出20倍的亲和力。停止流动紫外/可见研究和同位素标记实验共同认为，F21W突变既不明显地改变催化中间体的性质，也不会改变在DHP中为过氧化物酶和过氧酶活性建立的机制。 DHP B（F21W）的X射线晶体结构（1.96; PDB 5VLX）显示色氨酸嵌段两个鉴定的TCP结合位点之一，特别是TCP内部，表明其他部位TCPEXTERIOS对结合过氧环酶基材仍然可行。总之，这些研究表明，在其过氧化物酶活性的牺牲中选择性地封闭DHP中的TCP接触位点。

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