DszC protein

 

Dibenzothiophene (DBT) monooxygenase DszC is the key initiating enzyme in the 4S metabolic pathway, particularly found in the metabolism of many bacterial species, including Rhodococcus erythropolis, etc. DszC is responsible for the catalysis of the sequential sulphoxidation reaction of DBT to DBT sulfoxide (DBTO), then DBT sulfone (DBTO2).
Due to the detrimental effects of sulfur organic compounds found in crude oil, biodesulfurization involving DszC has gained great research interest as an alternative for the currently used hydrodesulfurization technique to produce cleaner fossil fuels at lower cost together with less CO2 emissions.

 

Reference Protein and Structure

Sequence
A0A0C6DRW4 UniProt IPR006091, IPR013107, IPR013786 (Sequence Homologues) (PDB Homologues)
Biological species
Rhodococcus erythropolis (Bacteria) Uniprot
PDB
3x0y - Crystal structure of FMN-bound DszC from Rhodococcus erythropolis D-1 (2.3 Å) PDBe PDBsum 3x0y
Catalytic CATH Domains
1.10.540.10 CATHdb 1.20.140.10 CATHdb 2.40.110.10 CATHdb (see all for 3x0y)
Click To Show Structure

Enzyme Reaction (EC:1.14.14.21)

FMNH2
CHEBI:16048ChEBI
+
singlet dioxygen
CHEBI:26689ChEBI
+
dibenzothiophene
CHEBI:23681ChEBI
→
FMN
CHEBI:17621ChEBI
+
dibenzothiophene 5-oxide
CHEBI:23683ChEBI
+
water
CHEBI:15377ChEBI
Alternative enzyme names: DszC (gene name),

Enzyme Mechanism

Introduction

Mechanistic studies have shown that sulfur oxidation of dibenzothiophenes (DBT) by DszC occurs in three major stages catalyzed by five main catalytic residues: His92, Tyr96, Asn129, Ser163, and His391. Firstly, Cys391 catalyzes molecular oxygen activation through proton-coupled electron transfer from FMNH2 to dioxygen, forming C4a-hydroperoxyflavin (C4aOOH) intermediate. Then, oxidation of DBT to DBTO is achieved via nucleophilic attack of DBT-sulfur atom to the distal oxygen atom in C4aOOH. Lastly, proton transfer N5 atom of C4aOOH intermediate to the His92-imidazole via the Ser163-hydroxyl and cleavage of the C4a−Op (Op = proximal oxygen atom) bond by Tyr96 collectively result in dehydration of FMN cofactor.

Catalytic Residues Roles

UniProt PDB* (3x0y)
His92 His92A His92 acts as an acid to deprotonate N5H in C4aOH flavin intermediate via Ser163 proton acceptor, proton donor
Tyr96 Tyr96A Tyr96 promotes DBT-sulfur oxidation by anchoring the proximal oxygen of C4a-hydroperoxyflavin (C4aOOH) and also acts as an acid for regeneration of oxidized FMN cofactor and a water molecule byproduct. proton acceptor, proton donor
Asn129 Asn129A Asn129 carbamide forms a hydrogen bond with the C4aOOH intermediate during dibenzothiophene (DBT) oxidation. hydrogen bond acceptor
His391 His391A His391 is a key residue that catalyzes the activation of molecular oxygen by FMNH2 cofactor, leading to the formation of the C4a-hydroperoxyflavin (C4aOOH) intermediate. His391 also forms a hydrogen bond to anchor OOH radical in a position close to the isoalloxazine of FMNH. hydrogen bond acceptor, proton acceptor, proton donor
Ser163 Ser163A Ser163 is first deprotonated by His92 to then deprotonate N5 atom in C4aOH intermediate, an important step required for further dehydration of C4aOH by Tyr96. hydrogen bond acceptor, proton acceptor, proton donor
His388 His388A His388 is a part of His triad (His92-His388-His391). From analysis, His388 might not very relevant in the catalysis by DszC, however, since the deletion of His388 leads to loss of enzymatic activity, His388 might be involved in the stabilization or binding of the substrates (inferred by the curator). electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, electron transfer, radical formation, colligation, intermediate formation, radical termination, bimolecular nucleophilic addition, overall product formed, dehydration

References

  1. Barbosa ACC et al. (2018), ACS Catal, 8, 9298-9311. Mechanistic Studies of a Flavin Monooxygenase: Sulfur Oxidation of Dibenzothiophenes by DszC. DOI:10.1021/acscatal.8b01877.
  2. Guan LJ et al. (2015), FEBS J, 282, 3126-3135. Crystal structures of apo-DszC and FMN-bound DszC from Rhodococcus erythropolis D-1. DOI:10.1111/febs.13216. PMID:25627402.
  3. Visitsatthawong S et al. (2015), J Am Chem Soc, 137, 9363-9374. Mechanism of Oxygen Activation in a Flavin-Dependent Monooxygenase: A Nearly Barrierless Formation of C4a-Hydroperoxyflavin via Proton-Coupled Electron Transfer. DOI:10.1021/jacs.5b04328. PMID:26144862.
  4. Zhang L et al. (2014), Proteins, 82, 2733-2743. Structural insights into the stabilization of active, tetrameric DszC by its C-terminus. DOI:10.1002/prot.24638. PMID:24975806.
  5. Liu S et al. (2014), Proteins, 82, 1708-1720. Crystal structure of DszC from Rhodococcus sp. XP at 1.79 Å. DOI:10.1002/prot.24525. PMID:24470304.

Step 1. His391 deprotonates FMNH2 cofactor, generating FMNH-

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Catalytic Residues Roles

Residue Roles
His391A proton acceptor
His388A electrostatic stabiliser

Chemical Components

proton transfer

Step 2. Proton-coupled electron transfer (PCET) from FMNH- to molecular oxygen and to C4a-carbon atom of the isoalloxazine ring, leading to formation of FMNH• and OOH• radicals.

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Catalytic Residues Roles

Residue Roles
His391A proton donor

Chemical Components

proton transfer, electron transfer, radical formation

Step 3. FMNH• and OOH• radicals covalently bond with each other in which each of the molecular fragments donate one of the bonding electrons, resulting in formation of C4a-hydroperoxyflavin (C4aOOH) intermediate.

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Catalytic Residues Roles

Residue Roles
His391A hydrogen bond acceptor

Chemical Components

colligation, intermediate formation, radical termination

Step 4. DBT sulfur atom performs a nucleophilic attack towards the distal oxygen atom (Od) of the C4aOOH intermediate. DBT (dibenzothiophene) is oxidized to DBTO (dibenzothiophene oxide) by following a SN2 mechanism.

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Catalytic Residues Roles

Residue Roles
His391A hydrogen bond acceptor
Asn129A hydrogen bond acceptor
Ser163A hydrogen bond acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, overall product formed

Step 5. Deprotonation of Ser163- hydroxyl group by the Nε atom of His92 residue.

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Catalytic Residues Roles

Residue Roles
Ser163A proton donor
His92A proton acceptor

Chemical Components

proton transfer

Step 6. Ser163 deprotonates the N5 of C4aOH intermediate.

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Catalytic Residues Roles

Residue Roles
Ser163A proton acceptor

Chemical Components

proton transfer

Step 7. Tyr96 promotes dehydration of C4aOH intermediate by cleavage of the C4a−Op bond. (Op = proximal oxygen atom) Generation of oxidized FMN cofactor.

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Catalytic Residues Roles

Residue Roles
Tyr96A proton donor

Chemical Components

dehydration, proton transfer

Step 8. Tyr96-phenoxide deprotonates the His92-imidazolium, allowing for regeneration of the original catalytic residues.

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Catalytic Residues Roles

Residue Roles
Tyr96A proton acceptor
His92A proton donor

Chemical Components

proton transfer
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Step 1. His391 deprotonates FMNH2 cofactor, generating FMNH-

Step 2. Proton-coupled electron transfer (PCET) from FMNH- to molecular oxygen and to C4a-carbon atom of the isoalloxazine ring, leading to formation of FMNH• and OOH• radicals.

Step 3. FMNH• and OOH• radicals covalently bond with each other in which each of the molecular fragments donate one of the bonding electrons, resulting in formation of C4a-hydroperoxyflavin (C4aOOH) intermediate.

Step 4. DBT sulfur atom performs a nucleophilic attack towards the distal oxygen atom (Od) of the C4aOOH intermediate. DBT (dibenzothiophene) is oxidized to DBTO (dibenzothiophene oxide) by following a SN2 mechanism.

Step 5. Deprotonation of Ser163- hydroxyl group by the Nε atom of His92 residue.

Step 6. Ser163 deprotonates the N5 of C4aOH intermediate.

Step 7. Tyr96 promotes dehydration of C4aOH intermediate by cleavage of the C4a−Op bond. (Op = proximal oxygen atom) Generation of oxidized FMN cofactor.

Step 8. Tyr96-phenoxide deprotonates the His92-imidazolium, allowing for regeneration of the original catalytic residues.

Products.

Contributors

Trung Nguyen, Marko Babić