Phosphonoacetaldehyde hydrolase

 

Phoshonoacetaldehyde hydrolase (phosphonatase) from Bacillus cereus catalyses the hydrolytic P-C bond cleavage of phosphonoacetaldehyde (Pald) to form orthophosphate and acetaldehyde. It is a process involved in metabolism in bacteria.

 

Reference Protein and Structure

Sequence
O31156 UniProt (3.11.1.1) IPR006323 (Sequence Homologues) (PDB Homologues)
Biological species
Bacillus cereus (Bacteria) Uniprot
PDB
1rql - Crystal Structure of Phosponoacetaldehyde Hydrolase Complexed with Magnesium and the Inhibitor Vinyl Sulfonate (2.4 Å) PDBe PDBsum 1rql
Catalytic CATH Domains
3.40.50.1000 CATHdb 1.10.150.240 CATHdb (see all for 1rql)
Cofactors
Magnesium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:3.11.1.1)

water
CHEBI:15377ChEBI
+
phosphonoacetaldehyde(1-)
CHEBI:58383ChEBI
→
acetaldehyde
CHEBI:15343ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: 2-phosphonoacetylaldehyde phosphonohydrolase, Phosphonatase, Phosphonoacetylaldehyde phosphonohydrolase,

Enzyme Mechanism

Introduction

This reaction proceeds via a Schiff-base intermediate. The nitrogen atom of side-chain Lys 53 nucleophilically attacks the carbonyl of Pald. The oxygen atom is then protonated by Lys 53. An extensive hydrogen bond network between the backbone carbonyl of Ala 45, Met 49, water and His 56 increases the basicity of His 56. A second nucleophilic attack by the N atom of Lys 53 creates C=N and a leaving group hydroxide which is protonated as it leaves by a water molecule, which in turn is protonated by His 56. The side-chain oxygen atom of the carbonyl of Asp 12 then nucleophilically attacks the P atom of the substrate, breaking the C-P bond, and pushing electrons up to the N atom of Lys 53. Nucleophilic attack of Lys 53 reforms the C=N bond, causing the C2 atom to deprotonate a water molecule, activating it. This water molecule then nucleophilically attacks P, breaking the P-OAsp 12 bond and forming the orthophosphate. His 56 deprotonates a water molecule, which in turn deprotonates a second water molecule, activating it for nucleophilic attack on the C1 atom, breaking the C=N bond. The hydroxyl now attached to C1 is then deprotonated by the N atom of Lys 53. The oxygen atom attached to C1 then nucleophilically attacks the C1 atom again, this time completely breaking the C-NLys 53 bond, forming the aldehyde.

Catalytic Residues Roles

UniProt PDB* (1rql)
Met46 Met49A An extensive hydrogen bond network between Met 49, Ala 45, water and His 56 serves to increase the basicity of His 56, and facilitate proton transfer. activator, hydrogen bond acceptor, electrostatic stabiliser
His53 His56A His 56 acts as an acid/base by donating a proton to a water molecule (leading to the protonation of a leaving group hydroxyl.) His 56 then deprotonates a water molecule, which in turn deprotonates a second water molecule, activating it for nucleophilic attack. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Arg157 Arg160A Stabilises the negative charge on the phosphate group hydrogen bond donor, electrostatic stabiliser
Ala11 (main-C), Asp183 Ala14A (main-C), Asp186A Forms part of the magnesium binding site. metal ligand
Ala42 (main-C) Ala45A (main-C) An extensive hydrogen bond network between the backbone carbonyl of Ala 45, Met 49, water and His 56 serves to increase the basicity of His 56, and facilitate proton transfer. hydrogen bond acceptor, electrostatic stabiliser
Asp9 Asp12A The side-chain carboxyl group of Asp 12 acts as a nucleophile by attacking the P atom of the substrate. Also forms part of the magnesium binding site. nucleophile, hydrogen bond acceptor, nucleofuge, metal ligand
Lys50 Lys53A Acts as a nucleophile and attacks the carbonyl of the substrate, and acts as an electrophile by accepting electrons from the carbon of C1. Also acts as an acid/base by initially protonating the oxygen atom attached to C1, then by deprotonating it at the end of the reaction. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleofuge, polar interaction, proton donor, proton acceptor, nucleophile, electron pair acceptor, electron pair donor
*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, enzyme-substrate complex formation, intermediate formation, proton transfer, unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, proton relay, dehydration, schiff base formed, bimolecular nucleophilic substitution, intermediate collapse, overall product formed, inferred reaction step, intermediate terminated, native state of enzyme regenerated

References

  1. Morais MC et al. (2004), J Biol Chem, 279, 9353-9361. X-ray Crystallographic and Site-directed Mutagenesis Analysis of the Mechanism of Schiff-base Formation in Phosphonoacetaldehyde Hydrolase Catalysis. DOI:10.1074/jbc.m312345200. PMID:14670958.
  2. Kamat SS et al. (2013), Curr Opin Chem Biol, 17, 589-596. The enzymatic conversion of phosphonates to phosphate by bacteria. DOI:10.1016/j.cbpa.2013.06.006. PMID:23830682.
  3. Lahiri SD et al. (2004), Biochemistry, 43, 2812-2820. Analysis of the Substrate Specificity Loop of the HAD Superfamily Cap Domain†,‡. DOI:10.1021/bi0356810. PMID:15005616.
  4. Zhang G et al. (2004), Biochemistry, 43, 4990-4997. Investigation of Metal Ion Binding in Phosphonoacetaldehyde Hydrolase Identifies Sequence Markers for Metal-Activated Enzymes of the HAD Enzyme Superfamily†,‡. DOI:10.1021/bi036309n. PMID:15109258.
  5. Morais MC et al. (2000), Biochemistry, 39, 10385-10396. The Crystal Structure ofBacillus cereusPhosphonoacetaldehyde Hydrolase:  Insight into Catalysis of Phosphorus Bond Cleavage and Catalytic Diversification within the HAD Enzyme Superfamily†,‡. DOI:10.1021/bi001171j.

Step 1. Lys53 attacks the carbonyl carbon of the phosphonoacetaldehyde aldehyde in a nucleophilic addition.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala14A (main-C) metal ligand
Asp12A metal ligand
Asp186A metal ligand
His56A hydrogen bond donor
Ala45A (main-C) hydrogen bond acceptor, electrostatic stabiliser
Met49A hydrogen bond acceptor, electrostatic stabiliser
Arg160A hydrogen bond donor, electrostatic stabiliser
Asp12A hydrogen bond acceptor
Lys53A polar interaction
Lys53A nucleophile

Chemical Components

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

Step 2. The oxyanion deprotonates the covalently attached Lys53.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala14A (main-C) metal ligand
Asp12A metal ligand
Asp186A metal ligand
His56A hydrogen bond donor
Ala45A (main-C) hydrogen bond acceptor, electrostatic stabiliser
Met49A hydrogen bond acceptor, electrostatic stabiliser
Arg160A hydrogen bond donor, electrostatic stabiliser
Asp12A hydrogen bond acceptor
Lys53A covalently attached, hydrogen bond donor
Lys53A proton donor

Chemical Components

proton transfer, intermediate formation

Step 3. Lys53 initiates an elimination that results in the loss of the alcohol group, which deprotonates a water which in turn deprotonates His56.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala14A (main-C) metal ligand
Asp12A metal ligand
Asp186A metal ligand
His56A hydrogen bond donor
Ala45A (main-C) hydrogen bond acceptor, electrostatic stabiliser
Met49A hydrogen bond acceptor, electrostatic stabiliser
Arg160A hydrogen bond donor, electrostatic stabiliser
Asp12A hydrogen bond acceptor
Lys53A covalently attached
His56A proton donor
Lys53A electron pair donor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, intermediate formation, proton relay, dehydration, schiff base formed

Step 4. Asp12 attacks the phosphate of the covalently bound intermediate in a nucleophilic substitution that cleaved the C-P bond and results in a phosphorylated Asp12 and alkylated Lys53

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His56A hydrogen bond acceptor, hydrogen bond donor
Ala45A (main-C) hydrogen bond acceptor
Met49A hydrogen bond acceptor
Arg160A hydrogen bond donor, electrostatic stabiliser
Asp12A hydrogen bond acceptor
Lys53A covalently attached
Ala14A (main-C) metal ligand
Asp12A metal ligand
Asp186A metal ligand
Asp12A nucleophile
Lys53A electron pair acceptor

Chemical Components

ingold: bimolecular nucleophilic substitution, enzyme-substrate complex formation, enzyme-substrate complex cleavage, intermediate collapse, intermediate formation

Step 5. Lys53 initiates a double bond rearrangement that causes the reduction of the C=C in the alkylated intermediate and deprotonates water. The activated water attacks the phosphate in a nucleophilic substitution that releases Asp12 and the phosphate product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His56A hydrogen bond acceptor, hydrogen bond donor
Ala45A (main-C) hydrogen bond acceptor
Met49A hydrogen bond acceptor
Arg160A hydrogen bond donor, electrostatic stabiliser
Asp12A hydrogen bond acceptor
Lys53A covalently attached
Ala14A (main-C) metal ligand
Asp12A metal ligand
Asp186A metal ligand
Asp12A nucleofuge
Lys53A electron pair donor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, overall reactant used, enzyme-substrate complex cleavage, intermediate collapse, intermediate formation, overall product formed

Step 6. His56 deprotonates water, which deprotonates a second water, which attacks the carbon of the C=N bond in a nucleophilic addition.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His56A hydrogen bond acceptor, hydrogen bond donor
Ala45A (main-C) hydrogen bond acceptor
Met49A hydrogen bond acceptor, activator
Arg160A hydrogen bond donor
Asp12A hydrogen bond acceptor
Lys53A covalently attached
Ala14A (main-C) metal ligand
Asp12A metal ligand
Asp186A metal ligand
His56A proton acceptor
Lys53A electron pair acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation, intermediate formation, proton relay, inferred reaction step

Step 7. Lys53 deprotonates the newly formed alcohol group.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His56A hydrogen bond donor
Ala45A (main-C) hydrogen bond acceptor, electrostatic stabiliser
Met49A hydrogen bond acceptor, electrostatic stabiliser
Arg160A hydrogen bond donor
Asp12A hydrogen bond acceptor
Lys53A covalently attached, hydrogen bond acceptor
Ala14A (main-C) metal ligand
Asp12A metal ligand
Asp186A metal ligand
Lys53A proton acceptor

Chemical Components

proton transfer, intermediate formation, inferred reaction step

Step 8. The oxyanion initiates an elimination that releases the acetaldehyde product and regenerates the active site Lys53.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His56A hydrogen bond donor
Ala45A (main-C) hydrogen bond acceptor, electrostatic stabiliser
Met49A hydrogen bond acceptor, electrostatic stabiliser
Arg160A hydrogen bond donor
Asp12A hydrogen bond acceptor
Lys53A covalently attached
Ala14A (main-C) metal ligand
Asp12A metal ligand
Asp186A metal ligand
Lys53A nucleofuge

Chemical Components

ingold: unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, intermediate terminated, intermediate collapse, overall product formed, native state of enzyme regenerated, inferred reaction step
Download: Image, Marvin File

Step 1. Lys53 attacks the carbonyl carbon of the phosphonoacetaldehyde aldehyde in a nucleophilic addition.

Step 2. The oxyanion deprotonates the covalently attached Lys53.

Step 3. Lys53 initiates an elimination that results in the loss of the alcohol group, which deprotonates a water which in turn deprotonates His56.

Step 4. Asp12 attacks the phosphate of the covalently bound intermediate in a nucleophilic substitution that cleaved the C-P bond and results in a phosphorylated Asp12 and alkylated Lys53

Step 5. Lys53 initiates a double bond rearrangement that causes the reduction of the C=C in the alkylated intermediate and deprotonates water. The activated water attacks the phosphate in a nucleophilic substitution that releases Asp12 and the phosphate product.

Step 6. His56 deprotonates water, which deprotonates a second water, which attacks the carbon of the C=N bond in a nucleophilic addition.

Step 7. Lys53 deprotonates the newly formed alcohol group.

Step 8. The oxyanion initiates an elimination that releases the acetaldehyde product and regenerates the active site Lys53.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Ellie Wright, Charity Hornby