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Hydroxyl Radical-Coupled Electron-Transfer Mechanism of Flavin-Dependent Hydroxylases.

ABSTRACT: Class A flavin-dependent hydroxylases (FdHs) catalyze the hydroxylation of organic compounds in a site- and stereoselective manner. In stark contrast, conventional synthetic routes require environmentally hazardous reagents and give modest yields. Thus, understanding the detailed mechanism of this class of enzymes is essential to their rational manipulation for applications in green chemistry and pharmaceutical production. Both electrophilic substitution and radical intermediate mechanisms have been proposed as interpretations of FdH hydroxylation rates and optical spectra. While radical mechanistic steps are often difficult to examine directly, modern quantum chemistry calculations combined with statistical mechanical approaches can yield detailed mechanistic models providing insights that can be used to differentiate reaction pathways. In the current work, we report quantum mechanical/molecular mechanical (QM/MM) calculations on the fungal TropB enzyme that shows an alternative reaction pathway in which hydroxylation through a hydroxyl radical-coupled electron-transfer mechanism is significantly favored over electrophilic substitution. Furthermore, QM/MM calculations on several modified flavins provide a more consistent interpretation of the experimental trends in the reaction rates seen experimentally for a related enzyme, para-hydroxybenzoate hydroxylase. These calculations should guide future enzyme and substrate design strategies and broaden the scope of biological spin chemistry.

SUBMITTER: Tweedy SE

PROVIDER: S-EPMC6943927 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Hydroxyl Radical-Coupled Electron-Transfer Mechanism of Flavin-Dependent Hydroxylases.

Tweedy Sara E SE Rodríguez Benítez Attabey A Narayan Alison R H ARH Zimmerman Paul M PM Brooks Charles L CL Wymore Troy T

The journal of physical chemistry. B 20190918 38

Class A flavin-dependent hydroxylases (FdHs) catalyze the hydroxylation of organic compounds in a site- and stereoselective manner. In stark contrast, conventional synthetic routes require environmentally hazardous reagents and give modest yields. Thus, understanding the detailed mechanism of this class of enzymes is essential to their rational manipulation for applications in green chemistry and pharmaceutical production. Both electrophilic substitution and radical intermediate mechanisms have ...[more]

PMID: 31532200

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Project description:UnlabelledShewanella oneidensis strain MR-1 is widely studied for its ability to respire a diverse array of soluble and insoluble electron acceptors. The ability to breathe insoluble substrates is defined as extracellular electron transfer and can occur via direct contact or by electron shuttling in S. oneidensis. To determine the contribution of flavin electron shuttles in extracellular electron transfer, a transposon mutagenesis screen was performed with S. oneidensis to identify mutants unable to secrete flavins. A multidrug and toxin efflux transporter encoded by SO_0702 was identified and renamed bfe (bacterial flavin adenine dinucleotide [FAD] exporter) based on phenotypic characterization. Deletion of bfe resulted in a severe decrease in extracellular flavins, while overexpression of bfe increased the concentration of extracellular flavins. Strains lacking bfe had no defect in reduction of soluble Fe(III), but these strains were deficient in the rate of insoluble Fe(III) oxide reduction, which was alleviated by the addition of exogenous flavins. To test a different insoluble electron acceptor, graphite electrode bioreactors were set up to measure current produced by wild-type S. oneidensis and the ?bfe mutant. With the same concentration of supplemented flavins, the two strains produced similar amounts of current. However, when exogenous flavins were not supplemented to bioreactors, bfe mutant strains produced significantly less current than the wild type. We have demonstrated that flavin electron shuttling accounts for ~75% of extracellular electron transfer to insoluble substrates by S. oneidensis and have identified the first FAD transporter in bacteria.ImportanceExtracellular electron transfer by microbes is critical for the geochemical cycling of metals, bioremediation, and biocatalysis using electrodes. A controversy in the field was addressed by demonstrating that flavin electron shuttling, not direct electron transfer or nanowires, is the primary mechanism of extracellular electron transfer employed by the bacterium Shewanella oneidensis. We have identified a flavin adenine dinucleotide transporter conserved in all sequenced Shewanella species that facilitates export of flavin electron shuttles in S. oneidensis. Analysis of a strain that is unable to secrete flavins demonstrated that electron shuttling accounts for ~75% of the insoluble extracellular electron transfer capacity in S. oneidensis.

Dataset Information

Hydroxyl Radical-Coupled Electron-Transfer Mechanism of Flavin-Dependent Hydroxylases.

Publications

Hydroxyl Radical-Coupled Electron-Transfer Mechanism of Flavin-Dependent Hydroxylases.

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