Adenylyl-sulfate reductase

 

Adenylylsulfate (adenosine 5'-phosphosulfate) reductase (APS reductase) from Archaeoglobus fulgidus catalyses the reversible reduction of APS to sulfite and AMP. This pathway provides sulfate which serves as a terminal electron acceptor of an anaerobic respiratory electron transfer chain. Electrons are also provided for anoxygenic photosynthesis and denitrification.

 

Reference Protein and Structure

Sequences
O28603 UniProt
O28604 UniProt IPR011803 (Sequence Homologues) (PDB Homologues)
Biological species
Archaeoglobus fulgidus DSM 4304 (Archaea) Uniprot
PDB
1jnr - Structure of adenylylsulfate reductase from the hyperthermophilic Archaeoglobus fulgidus at 1.6 resolution (1.6 Å) PDBe PDBsum 1jnr
Catalytic CATH Domains
3.50.50.60 CATHdb 3.30.70.20 CATHdb 3.90.700.10 CATHdb (see all for 1jnr)
Cofactors
Fadh2(2-) (1), Tetra-mu3-sulfido-tetrairon (2), Water (3) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:1.8.99.2)

5'-adenylyl sulfate(2-)
CHEBI:58243ChEBI
+
hydrogen donor
CHEBI:17499ChEBI
→
adenosine 5'-monophosphate(2-)
CHEBI:456215ChEBI
+
sulfite
CHEBI:17359ChEBI
+
hydron
CHEBI:15378ChEBI
+
acceptor
CHEBI:15339ChEBI
Alternative enzyme names: APS reductase, APS-reductase, Adenosine 5'-phosphosulfate reductase, Adenosine phosphosulfate reductase, AMP, sulfite:(acceptor) oxidoreductase (adenosine-5'-phosphosulfate-forming),

Enzyme Mechanism

Introduction

The two electrons required for APS reduction are transferred from an unknown electron donor, via two [4Fe-4S] clusters from the surface of the protein to FAD. Trp 48 aids in electron transfer between cluster I and the methyl C8M group of FAD. Once the FAD has been fully oxidised, a water molecule is made more basic by the hydrogen bond network of Asn 74, Glu 141 and Asp 361. This water then accepts a proton the N5 atom of reduced FAD, acting as a general base, activating N5 for nucleophilic attack on the sulphur atom of APS. A process that is assisted by Asn 74, Trp 234 and Arg 265, which act to make the sulphur atom more electrophilic by hydrogen bonding to the sulfate oxygens of APS. The process occurs via a negatively charged, penta-coordinated intermediate which decomposes, breaking the S-O bond, forming the leaving group AMP. The resulting negative charge is then pushed onto the N5 atom of FAD, causing the N-S bond to break, releasing SO3-, which is protonated by either His 398 or a protonated water molecule as it leaves.

Catalytic Residues Roles

UniProt PDB* (1jnr)
His398 His398A Helps to increase the electrophilicity of the sulphur atom of APS. Also protonates the sulfite leaving group. hydrogen bond donor, electrostatic stabiliser
Asn74 Asn74A Forms a hydrogen bond network with Glu 141 and Asp 361 which increases the basicity of the water molecule which deprotonates N5 of reduced FAD. Also helps to increase the electrophilicity of the sulphur atom of APS. hydrogen bond acceptor, hydrogen bond donor, activator, electrostatic stabiliser, increase electrophilicity
Asp361 Asp361A Forms a hydrogen bond network with Glu 141 and Asn 74 which increases the basicity of the water molecule which deprotonates N5 of reduced FAD. activator, hydrogen bond acceptor
Trp234, Arg265 Trp234A, Arg265A Helps to increase the electrophilicity of the sulphur atom of APS. increase electrophilicity, hydrogen bond acceptor, hydrogen bond donor
Ser449 Ser449A Stabilises negative charge on the FAD cofactor. hydrogen bond donor, electrostatic stabiliser
Trp48 Trp48B Transfers electrons between the S3 of cluster I and the methyl group C8M of FAD. single electron relay, van der waals interaction, single electron acceptor, single electron donor
Glu141 Glu141A Forms a hydrogen bond network with Asn 74 and Asp 361 which increases the basicity of the water molecule which deprotonates N5 of reduced FAD. activator, hydrogen bond acceptor
*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

electron transfer, proton transfer, overall reactant used, cofactor used, native state of cofactor regenerated, intermediate formation, electron relay, overall product formed, bimolecular nucleophilic addition, rate-determining step, unimolecular elimination by the conjugate base, intermediate terminated, intermediate collapse, native state of enzyme regenerated

References

  1. Schiffer A et al. (2006), Biochemistry, 45, 2960-2967. Reaction Mechanism of the Iron−Sulfur Flavoenzyme Adenosine-5‘-Phosphosulfate Reductase Based on the Structural Characterization of Different Enzymatic States†,‡. DOI:10.1021/bi0521689. PMID:16503650.
  2. Fritz G et al. (2002), Proc Natl Acad Sci U S A, 99, 1836-1841. Structure of adenylylsulfate reductase from the hyperthermophilic Archaeoglobus fulgidus at 1.6-A resolution. DOI:10.1073/pnas.042664399. PMID:11842205.

Step 1. A single electron is transferred from the donor, through two iron-sulfur clusters and Trp48B to FAD, with concomitant deprotonation of water.

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Catalytic Residues Roles

Residue Roles
Asn74A hydrogen bond acceptor, hydrogen bond donor
Glu141A hydrogen bond acceptor
Asp361A hydrogen bond acceptor
Ser449A hydrogen bond donor
Trp48B van der waals interaction, single electron relay, single electron acceptor, single electron donor

Chemical Components

electron transfer, proton transfer, overall reactant used, cofactor used, native state of cofactor regenerated, intermediate formation, electron relay

Step 2. A second single electron is transferred from the donor, through two iron-sulfur clusters and Trp48B to FAD forming the N1 anion form of reduced FAD.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn74A hydrogen bond acceptor, hydrogen bond donor
Glu141A hydrogen bond acceptor
Asp361A hydrogen bond acceptor
Ser449A hydrogen bond donor
Trp48B van der waals interaction, single electron relay, single electron acceptor, single electron donor

Chemical Components

electron transfer, cofactor used, native state of cofactor regenerated, intermediate formation, overall product formed, electron relay

Step 3.

The N5 of FAD initiates a nucleophilic attack on the sulfur of the sulfate group in an addition reaction.

The negative charge is delocalised primarily over the N1-C=O2 group but also over the entire isoalloxazine ring including atom N5. The counter-balancing positive charge necessary to maintain the unprotonated state is provided by two hydrogen bonds donated from the polypeptide to the O2 atom of FAD and by the large dipole of the 30 Angstroms long helix that is pointing with its N-terminus directly toward the N1 atom [PMID:11842205]. By inspection of the crystal structure we attribute the stabilisation role to the main-chain amide of Asn74 and to the side-chain of Ser449. Main-chain amide of Ser449 is an hydrogen bond donor to N1 of the isoalloxazine ring. The formation of the short-lived FAD-APS transition state is most likely the rate-limiting step [PMID:16503650].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn74A hydrogen bond donor, increase electrophilicity, hydrogen bond acceptor, electrostatic stabiliser
Glu141A hydrogen bond acceptor
Trp234A hydrogen bond donor, hydrogen bond acceptor, increase electrophilicity
Arg265A hydrogen bond donor, increase electrophilicity
Asp361A hydrogen bond acceptor
Ser449A hydrogen bond donor, electrostatic stabiliser
Trp48B van der waals interaction

Chemical Components

ingold: bimolecular nucleophilic addition, overall reactant used, cofactor used, intermediate formation, rate-determining step

Step 4.

Water deprotonates the N5 of FAD. One of the oxygen anions of sulfate eliminates the phosphate group.

It is possible that a firmly bound water molecule located 3.8 Angstroms away from the N5 atom acts as the proton acceptor. The basicity of this water molecule is significantly increased because of the hydrogen bond network comprising the side chain amide of Asn74 and the carboxylate side chains of Glu141 and Asp361 [PMID:11842205].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn74A hydrogen bond donor, activator, hydrogen bond acceptor, electrostatic stabiliser
Glu141A hydrogen bond acceptor, activator
Arg265A hydrogen bond donor, electrostatic stabiliser
Asp361A hydrogen bond acceptor, activator
Ser449A hydrogen bond donor, electrostatic stabiliser
Trp48B van der waals interaction

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, intermediate formation, overall product formed

Step 5. The N5 of FAD initiates the elimination of the sulfite with concomitant deprotonation of water.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn74A hydrogen bond donor, electrostatic stabiliser, hydrogen bond acceptor
Glu141A hydrogen bond acceptor
Trp234A hydrogen bond donor
Arg265A hydrogen bond donor
Asp361A hydrogen bond acceptor
His398A hydrogen bond donor, electrostatic stabiliser
Ser449A hydrogen bond donor, electrostatic stabiliser
Trp48B van der waals interaction

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, overall reactant used, intermediate terminated, intermediate collapse, native state of cofactor regenerated, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. A single electron is transferred from the donor, through two iron-sulfur clusters and Trp48B to FAD, with concomitant deprotonation of water.

Step 2. A second single electron is transferred from the donor, through two iron-sulfur clusters and Trp48B to FAD forming the N1 anion form of reduced FAD.

Step 3.

The N5 of FAD initiates a nucleophilic attack on the sulfur of the sulfate group in an addition reaction.

The negative charge is delocalised primarily over the N1-C=O2 group but also over the entire isoalloxazine ring including atom N5. The counter-balancing positive charge necessary to maintain the unprotonated state is provided by two hydrogen bonds donated from the polypeptide to the O2 atom of FAD and by the large dipole of the 30 Angstroms long helix that is pointing with its N-terminus directly toward the N1 atom [PMID:11842205]. By inspection of the crystal structure we attribute the stabilisation role to the main-chain amide of Asn74 and to the side-chain of Ser449. Main-chain amide of Ser449 is an hydrogen bond donor to N1 of the isoalloxazine ring. The formation of the short-lived FAD-APS transition state is most likely the rate-limiting step [PMID:16503650].

Step 4.

Water deprotonates the N5 of FAD. One of the oxygen anions of sulfate eliminates the phosphate group.

It is possible that a firmly bound water molecule located 3.8 Angstroms away from the N5 atom acts as the proton acceptor. The basicity of this water molecule is significantly increased because of the hydrogen bond network comprising the side chain amide of Asn74 and the carboxylate side chains of Glu141 and Asp361 [PMID:11842205].

Step 5. The N5 of FAD initiates the elimination of the sulfite with concomitant deprotonation of water.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid