Preparation and Characterization of the Favorskiiase Flavoprotein EncM and Its Distinctive Flavin-N5-Oxide Cofactor

Teufel, Robin

doi:10.1016/bs.mie.2018.01.036

Cited by 14 publications

(8 citation statements)

References 16 publications

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“…This model is consistent with three of the four known flavin-N5-oxide-utilizing enzymes. It does not include EncM, − and the full scope of nature’s use of this new flavin oxidation state has not yet been uncovered.…”

mentioning

confidence: 99%

Hexachlorobenzene Catabolism Involves a Nucleophilic Aromatic Substitution and Flavin-N5-Oxide Formation

Adak

Begley

2019

Biochemistry

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confidence: 99%

Hexachlorobenzene Catabolism Involves a Nucleophilic Aromatic Substitution and Flavin-N5-Oxide Formation

Adak

Begley

2019

Biochemistry

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“…The activation of dioxygen by several two-component monooxygenase enzymes has been proposed to occur through the formation of a flavin N5 oxygenating intermediate. ,− An N5-oxide is often formed as an intermediate in the reaction or as a final product. For the group C two-component monooxygenase YxeK, the enzyme utilizes an N5-peroxyflavin to salvage S -(2-succino)cysteine; however, a stable N5-oxide intermediate is not observed .…”

Section: Discussionmentioning

confidence: 99%

Shorter Alkanesulfonate Carbon Chains Destabilize the Active Site Architecture of SsuD for Desulfonation

et al. 2022

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Bacteria have evolved to utilize alternative organosulfur sources when sulfur is limiting. The SsuE/SsuD and MsuE/MsuD enzymes expressed when sulfur sources are restricted, are responsible for providing specific bacteria with sulfur in the form of alkanesulfonates. In this study, we evaluated why two structurally and functionally similar FMNH2-dependent monooxygenase enzymes (MsuD and SsuD) are needed for the acquisition of alkanesulfonates in some bacteria. In desulfonation assays, MsuD was able to utilize the entire range of alkanesulfonates (C1–C10). However, SsuD was not able to utilize smaller alkanesulfonate substrates. Interestingly, SsuD had a similar binding affinity for methanesulfonate (MES) (15 ± 1 μM) as MsuD (12 ± 1 μM) even though SsuD was not able to catalyze the desulfonation of the MES substrate. SsuD and MsuD showed decreased proteolytic susceptibility in the presence of FMNH2 with MES and octanesulfonate (OCS). Tighter loop closure was observed for the MsuD/FMNH2 complex with MES and OCS compared to SsuD under comparable conditions. Analysis of the SsuD/FMNH2/MES structure using accelerated molecular dynamics simulations found three different conformations for MES, demonstrating the instability of the bound structure. Even when MES was bound in a similar fashion to OCS within the active site, the smaller alkane chain resulted in a shift of FMNH2 so that it was no longer in a position to catalyze the desulfonation of MES. The active site of SsuD requires a longer alkane chain to maintain the appropriate architecture for desulfonation.

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“…The latter was based on the observation of an N 5 -oxide species in some flavin monooxygenases that cleave carbon-hetero bonds, such as DszA, 24 RutA, 27 HcbA, 67 and EncM. 68 The pathway through C 4a OOH is unviable due to the high barrier (above 30 kcal•mol −1 ) for the transfer of OH to DBTO 2 and the breaking of the C−S bond. Such a high barrier is likely due to the unavailability of redox species within the enzyme other than the FMN and dioxygen themselves, which render it unlikely for an OH transfer from C 4a OOH to the DBTO 2 substrate of DszA.…”

Section: ■ Conclusionmentioning

confidence: 99%

DszA Catalyzes C–S Bond Cleavage through N⁵–Hydroperoxyl Formation

Ferreira,

Neves,

Miranda

et al. 2024

J. Chem. Inf. Model.

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Due to its detrimental impact on human health and the environment, regulations demand ultralow sulfur levels on fossil fuels, in particular in diesel. However, current desulfurization techniques are expensive and cannot efficiently remove heteroaromatic sulfur compounds, which are abundant in crude oil and concentrate in the diesel fraction after distillation. Biodesulfurization via the four enzymes of the metabolic 4S pathway of the bacterium Rhodococcus erythropolis (DszA-D) is a possible solution. However, the 4S pathway needs to operate at least 500 times faster for industrial applicability, a goal currently pursued through enzyme engineering. In this work, we unveil the catalytic mechanism of the flavin monooxygenase DszA. Surprisingly, we found that this enzyme follows a recently proposed atypical mechanism that passes through the formation of an N 5 OOH intermediate at the re side of the cofactor, aided by a well-defined, predominantly hydrophobic O 2 pocket. Besides clarifying the unusual chemical mechanism of the complex DszA enzyme, with obvious implications for understanding the puzzling chemistry of flavin-mediated catalysis, the result is crucial for the rational engineering of DszA, contributing to making biodesulfurization attractive for the oil refining industry.

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Preparation and Characterization of the Favorskiiase Flavoprotein EncM and Its Distinctive Flavin-N5-Oxide Cofactor

Cited by 14 publications

References 16 publications

Hexachlorobenzene Catabolism Involves a Nucleophilic Aromatic Substitution and Flavin-N5-Oxide Formation

Hexachlorobenzene Catabolism Involves a Nucleophilic Aromatic Substitution and Flavin-N5-Oxide Formation

Shorter Alkanesulfonate Carbon Chains Destabilize the Active Site Architecture of SsuD for Desulfonation

DszA Catalyzes C–S Bond Cleavage through N⁵–Hydroperoxyl Formation

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Preparation and Characterization of the Favorskiiase Flavoprotein EncM and Its Distinctive Flavin-N5-Oxide Cofactor

Cited by 14 publications

References 16 publications

Hexachlorobenzene Catabolism Involves a Nucleophilic Aromatic Substitution and Flavin-N5-Oxide Formation

Hexachlorobenzene Catabolism Involves a Nucleophilic Aromatic Substitution and Flavin-N5-Oxide Formation

Shorter Alkanesulfonate Carbon Chains Destabilize the Active Site Architecture of SsuD for Desulfonation

DszA Catalyzes C–S Bond Cleavage through N5–Hydroperoxyl Formation

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DszA Catalyzes C–S Bond Cleavage through N⁵–Hydroperoxyl Formation