Engineering Rieske oxygenase activity one piece at a time

Brimberry, Marley; Garcia, Alejandro Arcadio; Liu, Jianxin; Tian, Jiayi; Bridwell‐Rabb, Jennifer

doi:10.1016/j.cbpa.2022.102227

Cited by 16 publications

(21 citation statements)

References 64 publications

(132 reference statements)

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“…The Rieske nonheme iron oxygenases, or Rieske oxygenases, use a Rieske-type [2Fe-2S] cluster and a mononuclear iron center to activate molecular oxygen. These enzymes typically facilitate the functionalization of C–H bonds − and are often recognized for their ability to catalyze monooxygenation and dioxygenation chemistry. Rieske oxygenases have also been shown to catalyze a handful of other divergent reactions including formylation, C–C bond cleavage, and carbocyclization .…”

Section: Introductionmentioning

confidence: 99%

“…Specifically, an NdmA variant with two active site substitutions and the NdmB loop sequence has markedly increased activity on the native NdmB substrate and no activity on the native NdmA substrate (Figure S1B). Additional studies demonstrated that the activity of two α 3 Rieske oxygenases, SxtT and GxtA, which catalyze monooxygenation reactions at the C12 and C11 positions of the saxitoxin scaffold, respectively, can be attributed to the cooperative work of three protein regions ,, (Figure S1B). The mutation of six residues distributed across the active site, substrate entrance tunnel, and flexible β13-to-β14 connecting loop of SxtT into their GxtA counterparts results in creation of an SxtT variant that fully exhibits the site selectivity, substrate scope, and substrate specificity of GxtA. ,, The tunnel-lining residues of SxtT direct the substrate into the active site where it is held by active site and loop residues in the correct orientation for catalysis. ,, …”

Section: Introductionmentioning

confidence: 99%

“…Rieske oxygenases have also been shown to catalyze a handful of other divergent reactions including formylation, C–C bond cleavage, and carbocyclization . These transformations are integral to the biosynthesis of medically, industrially, and environmentally relevant natural products and to the degradation of aromatic and polyaromatic hydrocarbons. − However, despite the valuable applications of this chemistry, remaining gaps in knowledge regarding the structure–function relationships in this class of enzymes have limited the practical use of Rieske oxygenases as biocatalysts.…”

Section: Introductionmentioning

confidence: 99%

“…The mutation of six residues distributed across the active site, substrate entrance tunnel, and flexible β13-to-β14 connecting loop of SxtT into their GxtA counterparts results in creation of an SxtT variant that fully exhibits the site selectivity, substrate scope, and substrate specificity of GxtA. 6,27,28 The tunnel-lining residues of SxtT direct the substrate into the active site where it is held by active site and loop residues in the correct orientation for catalysis. 6,27,28 Along with a structural analysis that revealed the presence of an analogous flexible loop and calculable substrate entrance tunnels in other structurally characterized α 3 Rieske oxygenases, these previous results suggest that residues found both inside and outside of the active site regions have a widespread influence on catalysis.…”

Section: ■ Introductionmentioning

confidence: 99%

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Leveraging a Structural Blueprint to Rationally Engineer the Rieske Oxygenase TsaM

et al. 2023

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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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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

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Leveraging a Structural Blueprint to Rationally Engineer the Rieske Oxygenase TsaM

et al. 2023

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“…These features have been suggested to be potential targets for enzyme engineering strategies to enhance the applicability of ROs (Figure 4). [182] Early studies on the engineerability of ROs were carried out in the context of the biodegradation of environmental pollutants. Site saturation mutagenesis of the α‐subunit BphA1 of the BPDO from Pseudomonas sp.…”

Section: Reactivities Of Rosmentioning

confidence: 99%

Rieske Oxygenases and Other Ferredoxin‐Dependent Enzymes: Electron Transfer Principles and Catalytic Capabilities

2023

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Enzymes that depend on sophisticated electron transfer via ferredoxins (Fds) exhibit outstanding catalytic capabilities, but despite decades of research, many of them are still not well understood or exploited for synthetic applications. This review aims to provide a general overview of the most important Fddependent enzymes and the electron transfer processes involved. While several examples are discussed, we focus in particular on the family of Rieske non-heme iron-dependent oxygenases (ROs). In addition to illustrating their electron transfer principles and catalytic potential, the current state of knowledge on structure-function relationships and the mode of interaction between the redox partner proteins is reviewed. Moreover, we highlight several key catalyzed transformations, but also take a deeper dive into their engineerability for biocatalytic applications. The overall findings from these case studies highlight the catalytic capabilities of these biocatalysts and could stimulate future interest in developing additional Fddependent enzyme classes for synthetic applications.

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An Optimized System for the Study of Rieske Oxygenase‐catalyzed Hydroxylation Reactions In vitro

Runda,

Kremser,

Özgen

et al. 2023

ChemCatChem

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Rieske non-heme iron oxygenases (ROs) are primarily known for their ability to catalyze the stereoselective formation of vicinal cis-diols in a single step, endowing valuable products for pharmaceutical and chemical applications. In addition, ROs can catalyze several other oxidation reactions with high regio-and stereoselectivity and typically broad substrate scope. Owing to their dependence on multicomponent electron transfer, the majority of synthetic applications of ROs relies on recombinant whole-cell catalysts. In this context, important properties of the multicomponent system that determine the catalytic efficiency, including electron transfer via redox partner proteins, stability and uncoupling, have been investigated to a lesser extent in recent years. Here, we show for one of the most prominent ROs, the cumene dioxygenase from Pseudomonas fluorescens IP01 (CDO) that by developing and optimizing an efficient in vitro system, high catalytic activities can be achieved. In addition, we highlight that an efficient and continuous supplementation of electrons to the oxygenase is required to sustain their catalytic activity, while uncoupling can be a major limitation in CDO efficiency and stability.

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Engineering Rieske oxygenase activity one piece at a time

Cited by 16 publications

References 64 publications

Leveraging a Structural Blueprint to Rationally Engineer the Rieske Oxygenase TsaM

Leveraging a Structural Blueprint to Rationally Engineer the Rieske Oxygenase TsaM

Rieske Oxygenases and Other Ferredoxin‐Dependent Enzymes: Electron Transfer Principles and Catalytic Capabilities

An Optimized System for the Study of Rieske Oxygenase‐catalyzed Hydroxylation Reactions In vitro

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