Alpha-D-ribose 1-methylphosphonate 5-phosphate C-P-lyase

 

This radical SAM (AdoMet) enzyme is part of the C-P lyase complex, which is responsible for processing phophonates into usable phosphate. The enzyme from the bacterium Escherichia coli can act on additional alpha-D-ribose phosphonate substrates with different substituents attached to the phosphonate phosphorus (e.g. alpha-D-ribose-1- (N-(phosphonomethyl)glycine)-5-phosphate and alpha-D-ribose-1-(2-N- acetamidomethylphosphonate)-5-phosphate).

 

Reference Protein and Structure

Sequence
P16688 UniProt (4.7.1.1) IPR010306 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
4xb6 - Structure of the E. coli C-P lyase core complex (1.7 Å) PDBe PDBsum 4xb6
Catalytic CATH Domains
(see all for 4xb6)
Cofactors
Tetra-mu3-sulfido-tetrairon (1)
Click To Show Structure

Enzyme Reaction (EC:4.7.1.1)

hydron
CHEBI:15378ChEBI
+
S-adenosyl-L-methionine zwitterion
CHEBI:59789ChEBI
+
alpha-D-ribose 1-methylphosphonate 5-phosphate(3-)
CHEBI:68686ChEBI
→
5'-deoxyadenosine
CHEBI:17319ChEBI
+
5-phosphonato-alpha-D-ribose cyclic-1,2-phosphate(3-)
CHEBI:68687ChEBI
+
L-methionine zwitterion
CHEBI:57844ChEBI
+
methane
CHEBI:16183ChEBI
Alternative enzyme names: PhnJ (gene name),

Enzyme Mechanism

Introduction

The reaction cascade is initiated by formation of a 5'-deoxyadenosyl radical. This intermediate abstracts the pro-R hydrogen from Gly 32 to form a glycyl radical. Hydrogen atom transfer from Cys 272 to the Gly 32 radical generates a thiyl radical on the side chain of Cys 272. This radical attacks the phosphonate moiety of the substrate to create a thiophosphonate radical intermediate. Homolytic C–P bond cleavage and hydrogen atom transfer from the original pro-S hydrogen of Gly 32 produces a thiophosphate intermediate, methane, and regenerates the radical intermediate at Gly 32. The ultimate product, PRcP, is formed by nucleophilic attack of the C2 hydroxyl on the covalent thiophosphate intermediate.

Catalytic Residues Roles

UniProt PDB* (4xb6)
Gly32 Gly32D Acts as a radical hydrogen atom proton/donor. hydrogen radical acceptor, hydrogen radical donor
Cys266, Cys244, Cys241 Cys266D, Cys244D, Cys241D Forms the iron-sulfur cluster binding site. metal ligand
Cys272 Cys272D Acts as a hydrogen atom acceptor/donor and also as a radical combinant and nucleofuge. hydrogen radical donor, covalently attached, nucleofuge, proton acceptor, radical combinant
*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

inferred reaction step, homolysis, electron transfer, overall reactant used, overall product formed, hydrogen transfer, bimolecular homolytic addition, unimolecular homolytic elimination, proton transfer, native state of enzyme regenerated, bimolecular nucleophilic substitution

References

  1. Kamat SS et al. (2013), Nature, 497, 132-136. The catalytic mechanism for aerobic formation of methane by bacteria. DOI:10.1038/nature12061. PMID:23615610.
  2. Seweryn P et al. (2015), Nature, 525, 68-72. Structural insights into the bacterial carbon-phosphorus lyase machinery. DOI:10.1038/nature14683. PMID:26280334.
  3. Kamat SS et al. (2011), Nature, 480, 570-573. Intermediates in the transformation of phosphonates to phosphate by bacteria. DOI:10.1038/nature10622. PMID:22089136.

Step 1. Initial formation of the 5'-deoxyadenosyl radical. The mechanism is assumed to be identical to other Radical SAM type proteins.

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Catalytic Residues Roles

Residue Roles
Cys241D metal ligand
Cys266D metal ligand
Cys244D metal ligand

Chemical Components

inferred reaction step, homolysis, electron transfer, overall reactant used, overall product formed

Step 2. The adenosyl radical abstracts a hydrogen atom from the catalytic glycine residue. The products of this activation step represent the ground state of this enzyme.

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Catalytic Residues Roles

Residue Roles
Cys241D metal ligand
Cys266D metal ligand
Cys244D metal ligand
Gly32D hydrogen radical donor

Chemical Components

hydrogen transfer, overall product formed

Step 3. The glycyl radical abstracts a hydrogen atom from the catalytic cysteine residue.

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Catalytic Residues Roles

Residue Roles
Cys241D metal ligand
Cys266D metal ligand
Cys244D metal ligand
Cys272D hydrogen radical donor
Gly32D hydrogen radical acceptor

Chemical Components

hydrogen transfer

Step 4. The cysteinyl residue attacks the phosphate group, forming a pentavalent intermediate.

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Catalytic Residues Roles

Residue Roles
Cys241D metal ligand
Cys266D metal ligand
Cys244D metal ligand
Cys272D radical combinant

Chemical Components

ingold: bimolecular homolytic addition, overall reactant used

Step 5. The pentavalent intermediate collapses, eliminating a methyl radical with concomitant abstraction of a hydrogen atom from the glycine residue.

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Catalytic Residues Roles

Residue Roles
Cys241D metal ligand
Cys266D metal ligand
Cys244D metal ligand
Cys272D covalently attached
Gly32D hydrogen radical donor

Chemical Components

ingold: unimolecular homolytic elimination, hydrogen transfer, overall product formed

Step 6. One of the hydroxyl groups of the ribose intermediate initiates a nucleophilic attack on the covalently bound phosphate to regenerate the cysteine residue and final product. The enzyme is now in a state that it can perform another round of catalysis.

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Catalytic Residues Roles

Residue Roles
Cys241D metal ligand
Cys266D metal ligand
Cys244D metal ligand
Cys272D proton acceptor, nucleofuge

Chemical Components

proton transfer, overall product formed, native state of enzyme regenerated, ingold: bimolecular nucleophilic substitution
Download: Image, Marvin File

Step 1. Initial formation of the 5'-deoxyadenosyl radical. The mechanism is assumed to be identical to other Radical SAM type proteins.

Step 2. The adenosyl radical abstracts a hydrogen atom from the catalytic glycine residue. The products of this activation step represent the ground state of this enzyme.

Step 3. The glycyl radical abstracts a hydrogen atom from the catalytic cysteine residue.

Step 4. The cysteinyl residue attacks the phosphate group, forming a pentavalent intermediate.

Step 5. The pentavalent intermediate collapses, eliminating a methyl radical with concomitant abstraction of a hydrogen atom from the glycine residue.

Step 6. One of the hydroxyl groups of the ribose intermediate initiates a nucleophilic attack on the covalently bound phosphate to regenerate the cysteine residue and final product. The enzyme is now in a state that it can perform another round of catalysis.

Products.

Contributors

Gemma L. Holliday