Leveraging a Structural Blueprint to Rationally Engineer the Rieske Oxygenase TsaM.
Leveraging a Structural Blueprint to Rationally Engineer the Rieske Oxygenase TsaM.
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DOI：
10.1021/acs.biochem.3c00150
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发表时间：
2023-06-06
期刊：
影响因子：
2.9
通讯作者：
Bridwell-Rabb, Jennifer
中科院分区：
文献类型：
--
作者：
Tian, Jiayi;Garcia, Alejandro Arcadio;Donnan, Patrick H.;Bridwell-Rabb, Jennifer
Rieske非血红素铁加氧酶使用两个金属分配中心，一个Rieske型[2Fe-2S]簇和一个单核铁中心，在广泛的底物上催化氧化反应。这些酶被微生物广泛用于降解环境污染物，并在无数具有工业意义的生物合成途径中建立复杂性。然而，尽管这种化学有价值，但对这类酶的结构-功能关系缺乏了解，这限制了我们合理重新设计、优化并最终开发这些酶的化学的能力。因此，在这项工作中，通过结合可用的结构信息和最先进的蛋白质建模工具，我们证明了可以针对三个“热点”区域来改变Rieske加氧酶p-甲苯磺酸甲基单加氧酶(TSAM)的位点选择性、底物偏好和底物范围。通过突变分布在三个蛋白质区域的6到10个残基，Tsam被设计成表现为香草酸单加氧酶(VANA)或麦草畏单加氧酶(DDMC)。这一工程壮举意味着TSAM被合理地设计成在芳香底物的间位和邻位催化氧化反应，而不是它所青睐的天然对位，并且TSAM被重新设计成在麦草畏上进行化学操作，麦草是一种酶本身不接受的底物。因此，这项工作有助于我们了解Rieske加氧酶类的结构-功能关系，并为未来这些金属酶的工程提供基本原理。
Rieske nonheme iron oxygenases use two metallocenters, a Rieske-type [2Fe-2S] cluster and a mononuclear iron center, to catalyze oxidation reactions on a broad range of substrates. These enzymes are widely used by microorganisms to degrade environmental pollutants and to build complexity in a myriad of biosynthetic pathways that are industrially interesting. However, despite the value of this chemistry, there is a dearth of understanding regarding the structure–function relationships in this enzyme class, which limits our ability to rationally redesign, optimize, and ultimately exploit the chemistry of these enzymes. Therefore, in this work, by leveraging a combination of available structural information and state-of-the-art protein modeling tools, we show that three “hotspot” regions can be targeted to alter the site selectivity, substrate preference, and substrate scope of the Rieske oxygenase p-toluenesulfonate methyl monooxygenase (TsaM). Through mutation of six to 10 residues distributed between three protein regions, TsaM was engineered to behave as either vanillate monooxygenase (VanA) or dicamba monooxygenase (DdmC). This engineering feat means that TsaM was rationally engineered to catalyze an oxidation reaction at the meta and ortho positions of an aromatic substrate, rather than its favored native para position, and that TsaM was redesigned to perform chemistry on dicamba, a substrate that is not natively accepted by the enzyme. This work thus contributes to unlocking our understanding of structure–function relationships in the Rieske oxygenase enzyme class and expands foundational principles for future engineering of these metalloenzymes.
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