Production of novel Rieske dioxygenase metabolites enabled by enzyme engineering

Osifalujo, Elizabeth A.; Rutkowski, Bailey N.; Satterwhite, Louis R.; Betts, Phillip C.; Nkosi, Angel K.; Froese, Jordan T.

doi:10.1039/d3cy00262d

Cited by 4 publications

(1 citation statement)

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“…Enzymes provide incredible opportunities for highly precise catalytic reactivity. 1–10 They drive a vast swath of different catalytic reactions, ranging from the synthesis of chiral alcohols to interfacial hydrolysis of ester bonds. 11,12 In general, the specificity of the reaction is precisely tuned to substrate structure, which is critically important for biological processes.…”

Section: Introductionmentioning

confidence: 99%

Identification of key active residues and solution conditions that affect peptide-catalyzed ester hydrolysis

Meerbott,

Knecht

2024

New J. Chem.

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Section: Introductionmentioning

confidence: 99%

Identification of key active residues and solution conditions that affect peptide-catalyzed ester hydrolysis

Meerbott,

Knecht

2024

New J. Chem.

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A High‐Throughput in Vitro Assay System for the Detection of the Enzymatic Dihydroxylation of Aliphatic Olefins

Rutkowski,

Talley,

Combs

et al. 2024

Adv Synth Catal

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Rieske dioxygenase enzymes can perform the cis‐dihydroxylation of aliphatic olefins, representing a potential green alternative to established methods of performing this important transformation. However, the activity of the natural enzymes in this context is low relative to their more well‐known activity in the cis‐dihydroxylation of aromatics. To enable the engineering of dioxygenase enzymes for improved activity in the dihydroxylation of aliphatic olefins, we have developed an assay system to detect the relevant diol metabolites produced from whole‐cell fermentation cultures. Optimization studies were carried out to maximize the sensitivity of the assay system, and its utility in the in vitro screening of enzyme variant libraries was demonstrated. The assay system was utilized in screening studies that identified Rieske dioxygenase variants with significantly improved activity in the dihydroxylation of aliphatic olefins relative to the wild‐type enzyme.

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Engineering of Rieske dioxygenase variants with improved cis‐dihydroxylation activity for benzoates

Betts,

Blakely,

Rutkowski

et al. 2024

Biotech & Bioengineering

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Rieske dioxygenases have a long history of being utilized as green chemical tools in the organic synthesis of high‐value compounds, due to their capacity to perform the cis‐dihydroxylation of a wide variety of aromatic substrates. The practical utility of these enzymes has been hampered however by steric and electronic constraints on their substrate scopes, resulting in limited reactivity with certain substrate classes. Herein, we report the engineering of a widely used member of the Rieske dioxygenase class of enzymes, toluene dioxygenase (TDO), to produce improved variants with greatly increased activity for the cis‐dihydroxylation of benzoates. Through rational mutagenesis and screening, TDO variants with substantially improved activity over the wild‐type enzyme were identified. Homology modeling, docking studies, molecular dynamics simulations, and substrate tunnel analysis were applied in an effort to elucidate how the identified mutations resulted in improved activity for this polar substrate class. These analyses revealed modification of the substrate tunnel as the likely cause of the improved activity observed with the best‐performing enzyme variants.

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Production of novel Rieske dioxygenase metabolites enabled by enzyme engineering

Abstract: Despite their popularity as enantioselective catalysts, the range of applications for Rieske dioxygenases has remained limited by their substrate scopes and selectivity. Herein, we report the development of dioxygenase variants...

Cited by 4 publications

References 77 publications

Identification of key active residues and solution conditions that affect peptide-catalyzed ester hydrolysis

Identification of key active residues and solution conditions that affect peptide-catalyzed ester hydrolysis

A High‐Throughput in Vitro Assay System for the Detection of the Enzymatic Dihydroxylation of Aliphatic Olefins

Engineering of Rieske dioxygenase variants with improved cis‐dihydroxylation activity for benzoates

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