Structural basis for regioselectivity and stereoselectivity of product formation by naphthalene 1,2-dioxygenase
Structural basis for regioselectivity and stereoselectivity of product formation by naphthalene 1,2-dioxygenase
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DOI：
10.1128/jb.00707-06
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发表时间：
2006-10-01
影响因子：
3.2
通讯作者：
Ramaswamy, S.
中科院分区：
文献类型：
--
作者：
Ferraro, Daniel J.;Okerlund, Adam L.;Ramaswamy, S.
Rieske加氧酶（RO）系统是两组分和三组分的酶系统，催化芳香族底物形成顺式二氢二醇。污染土壤中污染物的降解和手性合成子的生成一直是反渗透研究的重点。底物特异性和产物区域选择性和立体选择性已被证明在不同的ROs之间有所不同。虽然改变RO功能的定向进化方法在过去已经取得了成功，但由于缺乏对结构的理解，这些酶的合理工程仍然面临挑战。本文研究了假单胞菌菌株NCIB 9816-4 （NDO-O9816-4）中萘1,2-双加氧酶ph -352突变引起的结构变化。本文介绍了天然形式的ph -352- val突变体和与菲、蒽配合物的结构，以及野生型NDO-O9816-4与菲、蒽、3-硝基甲苯配合物的结构。菲在phe352 - val突变体活性位点的结合方向与野生型不同，而蒽在这两种酶的结合位置相似。观察到3-硝基甲苯的两种取向，即生产取向和非生产取向。这些取向有助于解释为什么NDO-O9816-4与硝基苯双加氧酶形成的产物不同。这些结构之间以及与其他已知底物结合的活性氧的比较表明，底物在活性位点结合的方向是产物区域选择性和立体选择性的主要决定因素。
Rieske oxygenase (RO) systems are two- and three-component enzyme systems that catalyze the formation of cis-dihydrodiols from aromatic substrates. Degradation of pollutants in contaminated soil and generation of chiral synthons have been the major foci of RO research. Substrate specificity and product regio- and stereoselectivity have been shown to vary between individual ROs. While directed evolution methods for altering RO function have been successful in the past, rational engineering of these enzymes still poses a challenge due to the lack of structural understanding. Here we examine the structural changes induced by mutation of Phe-352 in naphthalene 1,2-dioxygenase from Pseudomonas sp. strain NCIB 9816-4 (NDO-O9816-4). Structures of the Phe-352-Val mutant in native form and in complex with phenanthrene and anthracene, along with those of wild-type NDO-O9816-4 in complex with phenanthrene, anthracene, and 3-nitrotoluene, are presented. Phenanthrene was shown to bind in a different orientation in the Phe-352-Val mutant active site from that in the wild type, while anthracene was found to bind in similar positions in both enzymes. Two orientations of 3-nitrotoluene were observed, i.e., a productive and a nonproductive orientation. These orientations help explain why NDO-O9816-4 forms different products from 3-nitrotoluene than those made from nitrobenzene dioxygenase. Comparison of these structures among themselves and with other known ROs bound to substrates reveals that the orientation of substrate binding at the active site is the primary determinant of product regio- and stereoselectivity.