Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA.
Biochemistry. 2013 Apr 2;52(13):2218-27. doi: 10.1021/bi301509r. Epub 2013 Mar 22.
Dehaloperoxidase (DHP A), a unique multifunctional enzyme, from the marine annelid Amphitrite ornata dehalogenates 2,4,6-tribromophenol to form 2,6-dibromo-1,4-benzoquinone. The catalytic cycle of DHP is similar to that of horseradish peroxidase (HRP), involving a high-valent ferryl heme and two single-electron transfers from the aromatic substrate to the enzyme. Like HRP, DHP has been investigated as a potential bioremediation enzyme. However, DHP fails as a bioremediation enzyme because, unlike HRP, it has an internal binding cavity on the distal side of the heme capable of accommodating p-bromophenols, which act as an inhibitor of peroxidase function. Blocking internal binding in DHP may be the key to allowing the enzyme to function effectively as a peroxidase on the full range of halogenated phenols. The distal cavity of DHP is surrounded by several hydrophobic amino acids that stabilize internal binding of the monohalogenated phenols, including a leucine residue near the back edge of the heme (L100). We have expressed the L100F, L100Q, L100T, and L100V mutants of DHP in an effort to prevent internal binding and thereby convert the inhibitors into substrates. Kinetic assays and resonance Raman indicate that the peroxidase activity of the L100T and L100F mutants is increased compared to that of native DHP in the presence of 4-bromophenol (4-BP), suggesting a reduction in the inhibitor binding constant. In addition, the X-ray crystal structure of L100F clearly indicates a reduced occupancy of the 4-BP inhibitor in the distal cavity of DHP. However, at the same time, the L100F structure reveals that steric interference alone is insufficient to exclude the inhibitor. Instead, the comparison of L100T and isosteric L100V reveals that an increase in polarity plays a decisive role in excluding the inhibitor from the distal binding pocket.
脱卤过氧化物酶（DHP A）是一种独特的多功能酶，来源于海洋环节动物 Amphitrite ornata，它能将 2,4,6-三溴苯酚脱卤形成 2,6-二溴-1,4-苯醌。DHP 的催化循环与辣根过氧化物酶（HRP）相似，涉及高价铁过氧化物血红素和两个来自芳香族底物的单电子转移到酶上。与 HRP 一样，DHP 已被研究作为一种潜在的生物修复酶。然而，DHP 不能作为一种生物修复酶，因为与 HRP 不同，它在血红素的远端有一个内部结合腔，能够容纳 p-溴苯酚，而 p-溴苯酚是过氧化物酶功能的抑制剂。阻止 DHP 中的内部结合可能是使该酶在全范围卤代酚类物质中有效作为过氧化物酶发挥作用的关键。DHP 的远端腔由几个疏水性氨基酸环绕，这些氨基酸稳定单卤代酚的内部结合，包括血红素后边缘附近的一个亮氨酸残基（L100）。我们已经在表达 L100F、L100Q、L100T 和 L100V 突变体的 DHP 中表达了 L100F、L100Q、L100T 和 L100V 突变体，以防止内部结合，从而将抑制剂转化为底物。动力学分析和共振拉曼表明，与天然 DHP 相比，L100T 和 L100F 突变体在 4-溴苯酚（4-BP）存在下的过氧化物酶活性增加，表明抑制剂结合常数降低。此外，L100F 的 X 射线晶体结构清楚地表明，在 DHP 的远端腔中，4-BP 抑制剂的占有率降低。然而，与此同时，L100F 结构表明，仅仅是空间位阻不足以将抑制剂排除在远端结合口袋之外。相反，L100T 和等立体 L100V 的比较表明，极性的增加在将抑制剂从远端结合口袋中排除方面起着决定性的作用。
