O-phospho-L-seryl-tRNASec:L-selenocysteinyl-tRNA synthase

 

A pyridoxal-phosphate dependent protein. Catalyses the conversion of O-phosphoseryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec), part of the selenoprotein biosynthesis pathway. In archaea and eukarya, selenocysteine formation is achieved in a two-step process where O-phosphoseryl-tRNASec kinase phosphorylates the endogenous L-seryl-tRNASec to O-phospho-L-seryl-tRNASec, followed by the conversion of the mis-acylated amino acid-tRNA species to L-selenocysteinyl-tRNASec by Sep-tRNA:Sec-tRNA synthase. The active site is formed at the dimer interface of the tetrameric units, with residues necessary for catalysis originating from both chains.

 

Reference Protein and Structure

Sequence
Q6LZM9 UniProt (2.9.1.2) IPR019872 (Sequence Homologues) (PDB Homologues)
Biological species
Methanococcus maripaludis S2 (Archaea) Uniprot
PDB
2z67 - Crystal structure of archaeal O-phosphoseryl-tRNA(Sec) selenium transferase (SepSecS) (2.5 Å) PDBe PDBsum 2z67
Catalytic CATH Domains
3.40.640.10 CATHdb (see all for 2z67)
Cofactors
Pyridoxal 5'-phosphate(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:2.9.1.2)

selenophosphate
CHEBI:16144ChEBI
+
water
CHEBI:15377ChEBI
+
3'-(O-phosphonato-L-seryl)adenylyl(2-) group
CHEBI:78551ChEBI
→
3'-(L-selenocysteinyl)adenylyl(1-) group
CHEBI:78573ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
Alternative enzyme names: MMPSepSecS, SepSecS, SLA/LP, O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase, O-phospho-L-seryl-tRNA:L-selenocysteinyl-tRNA synthase, O-phosphoseryl-tRNA(Sec) selenium transferase,

Enzyme Mechanism

Introduction

The amino group of tRNA-SEC attacks the Schiff base immine, forming a substrate-cofactor-enzyme intermediate. A proton is transferred from the tRNA-SEC nitrogen to that of the Lys278. An external aldamine adduct is formed between the tRNA-SEC substrate and PLP cofactor on elimination of Lys278. Lys278 abstracts a proton from the C(a) of the external aldimine. Electron delocalisation across the aromatic ring facilitates rapid beta-elimination of phosphate, forming dehydroalanyl-tRNASec as the product. An activated water molecule attacks the selenophosphate phosphate substrate, resulting in displacement if selenium which attacks the dehydroalanyl intermediate. A proton is donated to the intermediate from Lys278. Lys278 attacks the external aldimine. An internal proton rearrangement occurs. The side chain nitrogen of Lys278 displaces the anionic form of L-Selenocysteinyl-tRNA(Sec), reforming the internal aldimine.

Catalytic Residues Roles

UniProt PDB* (2z67)
Lys278 Lys278(298)A Covalently attached to the PLP cofactor in the ground state of the enzyme. Acts as a general acid/base as well as a catalytic nucleophile during the course of the reaction. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleofuge, polar interaction, proton acceptor, proton donor, nucleophile, activator, electron pair acceptor, electron pair donor
His166, Arg72, Ser95, Gln102, Ala249, Arg307, Arg94 His166(186)A, Arg72(92)B, Ser95(115)B, Gln102(122)B, Ala249(269)A, Arg307(327)B, Arg94(114)B These residues form part of the PLP-binding pocket and help to stabilise the reactive intermediates formed during the course of the reaction. electrostatic stabiliser, polar interaction
*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

bimolecular nucleophilic addition, overall reactant used, intermediate formation, enzyme-substrate complex formation, proton transfer, intramolecular elimination, enzyme-substrate complex cleavage, schiff base formed, charge delocalisation, overall product formed, cofactor used, bimolecular nucleophilic substitution, hydrolysis, intermediate terminated, inferred reaction step, native state of enzyme regenerated, intermediate collapse

References

  1. Palioura S et al. (2009), Science, 325, 321-325. The Human SepSecS-tRNASec Complex Reveals the Mechanism of Selenocysteine Formation. DOI:10.1126/science.1173755. PMID:19608919.
  2. Itoh Y et al. (2014), J Mol Biol, 426, 1723-1735. Dimer-dimer interaction of the bacterial selenocysteine synthase SelA promotes functional active-site formation and catalytic specificity. DOI:10.1016/j.jmb.2014.01.003. PMID:24456689.
  3. Araiso Y et al. (2008), Nucleic Acids Res, 36, 1187-1199. Structural insights into RNA-dependent eukaryal and archaeal selenocysteine formation. DOI:10.1093/nar/gkm1122. PMID:18158303.

Step 1. The amino group of tRNA-SEC attacks the Schiff base immine, forming a substrate-cofactor-enzyme intermediate.

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Catalytic Residues Roles

Residue Roles
Ala249(269)A steric role, van der waals interaction
Arg72(92)B hydrogen bond donor
Ser95(115)B hydrogen bond donor, electrostatic stabiliser
His166(186)A electrostatic stabiliser, polar interaction
Arg94(114)B hydrogen bond donor
Gln102(122)B hydrogen bond donor, electrostatic stabiliser
Arg307(327)B attractive charge-charge interaction, electrostatic stabiliser
Lys278(298)A activator, covalently attached
Lys278(298)A electron pair acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, overall reactant used, intermediate formation, enzyme-substrate complex formation

Step 2. A proton is transferred from the tRNA-SEC nitrogen to that of the Lys278.

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Catalytic Residues Roles

Residue Roles
Ala249(269)A steric role, van der waals interaction
Arg72(92)B hydrogen bond donor
Ser95(115)B hydrogen bond donor, electrostatic stabiliser
His166(186)A electrostatic stabiliser, polar interaction
Arg94(114)B hydrogen bond donor
Gln102(122)B hydrogen bond donor, electrostatic stabiliser
Arg307(327)B attractive charge-charge interaction, electrostatic stabiliser
Lys278(298)A covalently attached, hydrogen bond acceptor
Lys278(298)A proton acceptor

Chemical Components

proton transfer

Step 3. An external aldamine adduct is formed between the tRNA-SEC substrate and PLP cofactor on elimination of Lys278.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala249(269)A van der waals interaction, steric role
Arg72(92)B hydrogen bond donor
Ser95(115)B hydrogen bond donor, electrostatic stabiliser
His166(186)A electrostatic stabiliser, polar interaction
Arg94(114)B hydrogen bond donor
Gln102(122)B hydrogen bond acceptor, electrostatic stabiliser
Arg307(327)B attractive charge-charge interaction, electrostatic stabiliser
Lys278(298)A nucleofuge

Chemical Components

ingold: intramolecular elimination, enzyme-substrate complex cleavage, schiff base formed

Step 4. Lys278 abstracts a proton from the C(a) of the external aldimine.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala249(269)A van der waals interaction, steric role
Arg72(92)B hydrogen bond donor
Ser95(115)B hydrogen bond donor, electrostatic stabiliser
His166(186)A electrostatic stabiliser, polar interaction
Arg94(114)B hydrogen bond donor
Gln102(122)B hydrogen bond donor, electrostatic stabiliser
Arg307(327)B attractive charge-charge interaction, electrostatic stabiliser
Lys278(298)A activator, hydrogen bond acceptor
Lys278(298)A proton acceptor

Chemical Components

proton transfer, charge delocalisation, intermediate formation

Step 5. Electron delocalization across the aromatic ring facilitates rapid beta-elimination of phosphate, forming dehydroalanyl-tRNASec as the product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala249(269)A steric role, van der waals interaction
Arg72(92)B attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond donor
Ser95(115)B hydrogen bond donor, electrostatic stabiliser
His166(186)A electrostatic stabiliser, polar interaction
Arg94(114)B attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond donor
Gln102(122)B hydrogen bond donor, electrostatic stabiliser
Arg307(327)B attractive charge-charge interaction, electrostatic stabiliser
Lys278(298)A hydrogen bond donor

Chemical Components

ingold: intramolecular elimination, overall product formed, intermediate formation, cofactor used

Step 6. An activated water molecule attacks the selenophosphate phosphate substrate, resulting in displacement if selenium which attacks the dehydroalanyl intermediate. A proton is donated to the intermediate from Lys278.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala249(269)A van der waals interaction, steric role
Arg72(92)B attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond donor
Ser95(115)B hydrogen bond donor, electrostatic stabiliser
His166(186)A electrostatic stabiliser, polar interaction
Arg94(114)B attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond donor
Gln102(122)B hydrogen bond donor, electrostatic stabiliser
Arg307(327)B electrostatic stabiliser, attractive charge-charge interaction, hydrogen bond donor
Lys278(298)A activator, hydrogen bond donor
Lys278(298)A proton donor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, overall reactant used, intermediate formation, hydrolysis

Step 7. Lys278 attacks the external aldimine.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala249(269)A van der waals interaction, steric role
Arg72(92)B attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond donor
Ser95(115)B electrostatic stabiliser, hydrogen bond donor
His166(186)A electrostatic stabiliser, polar interaction
Arg94(114)B attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond donor
Gln102(122)B hydrogen bond donor, electrostatic stabiliser
Arg307(327)B attractive charge-charge interaction, electrostatic stabiliser
Lys278(298)A polar interaction, activator
Lys278(298)A nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation, enzyme-substrate complex formation

Step 8. An internal proton rearrangement occurs.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala249(269)A steric role, van der waals interaction
Arg72(92)B electrostatic stabiliser, attractive charge-charge interaction, hydrogen bond donor
Ser95(115)B electrostatic stabiliser, hydrogen bond donor
His166(186)A electrostatic stabiliser, polar interaction
Arg94(114)B electrostatic stabiliser, attractive charge-charge interaction, hydrogen bond donor
Gln102(122)B electrostatic stabiliser, hydrogen bond donor
Arg307(327)B electrostatic stabiliser, hydrogen bond donor, attractive charge-charge interaction
Lys278(298)A covalently attached, hydrogen bond donor
Lys278(298)A proton donor

Chemical Components

proton transfer, intermediate terminated, inferred reaction step

Step 9. The side chain nitrogen of Lys278 displaces the anionic form of L-Selenocysteinyl-tRNA(Sec), reforming the internal aldimine.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala249(269)A steric role, van der waals interaction
Arg72(92)B hydrogen bond donor
Ser95(115)B electrostatic stabiliser, hydrogen bond donor
His166(186)A electrostatic stabiliser, polar interaction
Arg94(114)B hydrogen bond donor
Gln102(122)B electrostatic stabiliser, hydrogen bond donor
Arg307(327)B electrostatic stabiliser, attractive charge-charge interaction, hydrogen bond donor
Lys278(298)A covalently attached
Lys278(298)A electron pair donor

Chemical Components

ingold: intramolecular elimination, native state of enzyme regenerated, overall product formed, intermediate collapse

Step 10. The terminal amine group deprotonates the unidentified base. The unidentified base is thought to correspond to an active site arginine residue, as seen in related mechanisms [PMID:18158303].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala249(269)A van der waals interaction, steric role
Arg72(92)B hydrogen bond donor
Ser95(115)B hydrogen bond donor, electrostatic stabiliser
His166(186)A electrostatic stabiliser, polar interaction
Arg94(114)B hydrogen bond donor
Gln102(122)B hydrogen bond donor, electrostatic stabiliser
Arg307(327)B attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond donor
Lys278(298)A covalently attached

Chemical Components

proton transfer, native state of enzyme regenerated, inferred reaction step
Download: Image, Marvin File

Step 1. The amino group of tRNA-SEC attacks the Schiff base immine, forming a substrate-cofactor-enzyme intermediate.

Step 2. A proton is transferred from the tRNA-SEC nitrogen to that of the Lys278.

Step 3. An external aldamine adduct is formed between the tRNA-SEC substrate and PLP cofactor on elimination of Lys278.

Step 4. Lys278 abstracts a proton from the C(a) of the external aldimine.

Step 5. Electron delocalization across the aromatic ring facilitates rapid beta-elimination of phosphate, forming dehydroalanyl-tRNASec as the product.

Step 6. An activated water molecule attacks the selenophosphate phosphate substrate, resulting in displacement if selenium which attacks the dehydroalanyl intermediate. A proton is donated to the intermediate from Lys278.

Step 7. Lys278 attacks the external aldimine.

Step 8. An internal proton rearrangement occurs.

Step 9. The side chain nitrogen of Lys278 displaces the anionic form of L-Selenocysteinyl-tRNA(Sec), reforming the internal aldimine.

Step 10. The terminal amine group deprotonates the unidentified base. The unidentified base is thought to correspond to an active site arginine residue, as seen in related mechanisms [PMID:18158303].

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday, Charity Hornby