Phytoene desaturase (lycopene-forming)

 

CRTI-type phytoene desaturase is an enzyme present in bacteria and fungi. The main role of the enzyme is the conversion of cis-phytoene, a 40-carbon molecule with a single cis bond, into all-trans lycopene. This reaction is a part of carotenoid synthesis. The CRTI gene encoding the enzyme is researched because of its importance in fortifying foods with provitamin A, for example Golden Rice, used to combat vitamin A malnutrition.

 

Reference Protein and Structure

Sequence
P21685 UniProt (1.3.99.31) IPR014105 (Sequence Homologues) (PDB Homologues)
Biological species
Pantoea ananatis (Bacteria) Uniprot
PDB
4dgk - Crystal structure of Phytoene desaturase CRTI from Pantoea ananatis (2.35 Å) PDBe PDBsum 4dgk
Catalytic CATH Domains
3.50.50.60 CATHdb (see all for 4dgk)
Cofactors
Fadh2(2-) (4)
Click To Show Structure

Enzyme Reaction (EC:1.3.99.31)

15-cis-phytoene
CHEBI:27787ChEBI
+
FAD
CHEBI:16238ChEBI
→
lycopene
CHEBI:15948ChEBI
+
FADH2
CHEBI:17877ChEBI
Alternative enzyme names: 4-step phytoene desaturase, Four-step phytoene desaturase, Phytoene desaturase, CrtI,

Enzyme Mechanism

Introduction

This reaction is FAD-dependent. It begins with Arg152 and Arg148 polarising a C-C double bond on the substrate and acidifying the neighbouring saturated carbons, facilitating the dehydrogenation. Dehydrogenation starts by Asp149 accepting a hydrogen and FAD accepting a hydride from the substrate. Asp149 then donates a proton to a cis double bond and creates a positive charge, stabilised by FADH-. The dehydrogenation step repeats four times and the isomerisation step once. It is not known after which dehydrogenation the isomerisation occurs. Here, is its shown in the first step for simplicity.

Catalytic Residues Roles

UniProt PDB* (4dgk)
Asp149 Asp149A Asp149 removes a proton from the substrate allowing the hydride to be abstracted and bound by the FAD. Asp149 also acts as a proton shuttle and lends its proton to a substrates cis double bond in order to rotate it into trans conformation. The protonated Asp149 is supposedly deprotonated by the negatively charged FADH-, but this is inferred by the curator. proton acceptor, proton donor
Arg152 Arg152A Arg152 polarizes a double bond acidifying the neighboring saturated carbons and priming them for dehydrogenation. increase acidity
Arg148 Arg148A Arg148 acidifies the C-H bond by positively polarizing the neighboring double bonds along with Arg152. increase acidity
Glu31 Glu31A Glu31 is essential for anchoring the FAD molecule to the active site. Glu31 creates hydrogen bonds with the ribose on the FAD molecule. 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

redox reaction, hydride transfer, proton transfer, intramolecular rearrangement, bond polarisation, atom stereo change, inferred reaction step, overall reactant used, overall product formed

References

  1. Schaub P et al. (2012), PLoS One, 7, e39550-. On the structure and function of the phytoene desaturase CRTI from Pantoea ananatis, a membrane-peripheral and FAD-dependent oxidase/isomerase. DOI:10.1371/journal.pone.0039550. PMID:22745782.
  2. Fraser PD et al. (1992), J Biol Chem, 267, 19891-19895. Expression in Escherichia coli, purification, and reactivation of the recombinant Erwinia uredovora phytoene desaturase. PMID:1400305.

Step 1. Arg152 and Arg148 polarise the C = C double bond, thereby acidifying the neighbouring C-H functionality. Asp149 abstracts a proton from the substrate, facilitating a hydride transfer to FAD from the substrate and forms a C-C double bond. Following this, a cis to trans isomerisation occurs in the substrate.

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Catalytic Residues Roles

Residue Roles
Asp149A proton acceptor
Glu31A electrostatic stabiliser
Arg152A increase acidity
Arg148A increase acidity

Chemical Components

redox reaction, hydride transfer, proton transfer, intramolecular rearrangement, bond polarisation, atom stereo change

Step 2. FADH- accepts a proton to stabilise the negative charge following hydride addition. FADH2 then leaves the active site and is replaced by a new FAD while also regenerating the native state of the enzyme. This step is inferred by the curator.

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Catalytic Residues Roles

Residue Roles
Asp149A proton donor

Chemical Components

proton transfer, inferred reaction step

Step 3. Asp149 deprotonates a saturated bond, allowing FAD to abstract a hydride. Arg152 and Arg148 acidify the saturated bond while Glu31 binds the FAD in place.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp149A proton acceptor
Glu31A electrostatic stabiliser
Arg152A increase acidity
Arg148A increase acidity

Chemical Components

bond polarisation, proton transfer, hydride transfer, redox reaction, intramolecular rearrangement

Step 4. FADH- accepts a proton to stabilise the negative charge following hydride addition. FADH2 then leaves the active site and is replaced by a new FAD while also regenerating the native state of the enzyme. This step is inferred by the curator.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp149A proton donor

Chemical Components

proton transfer, inferred reaction step

Step 5. Asp149 removes a proton from a saturated bond, allowing FAD to abstract a hydride. Arg152 and Arg148 acidify the saturated bond while Glu31 binds the FAD in place using electrostatic interactions.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp149A proton acceptor
Arg152A increase acidity
Glu31A electrostatic stabiliser
Arg148A increase acidity

Chemical Components

bond polarisation, proton transfer, hydride transfer, intramolecular rearrangement, redox reaction

Step 6. FADH- accepts a proton to stabilise the negative charge following hydride addition. FADH2 then leaves the active site and is replaced by a new FAD while also regenerating the native state of the enzyme.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu31A electrostatic stabiliser
Asp149A proton donor

Chemical Components

proton transfer

Step 7. Arg152 and Arg148 polarise the C = C double bond, thereby acidifying the neighbouring C-H functionality. Asp149 abstracts a proton from the substrate, facilitating a hydride transfer to FAD from the substrate and forms a C-C double bond.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp149A proton acceptor
Arg152A increase acidity
Glu31A electrostatic stabiliser
Arg148A increase acidity

Chemical Components

proton transfer, hydride transfer, bond polarisation, redox reaction, intramolecular rearrangement

Step 8. FADH- accepts a proton to stabilise the negative charge following hydride addition. FADH2 then leaves the active site and is replaced by a new FAD while also regenerating the native state of the enzyme. This step is inferred by the curator.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp149A proton donor

Chemical Components

proton transfer, overall reactant used, overall product formed, inferred reaction step
Download: Image, Marvin File

Step 1. Arg152 and Arg148 polarise the C = C double bond, thereby acidifying the neighbouring C-H functionality. Asp149 abstracts a proton from the substrate, facilitating a hydride transfer to FAD from the substrate and forms a C-C double bond. Following this, a cis to trans isomerisation occurs in the substrate.

Step 2. FADH- accepts a proton to stabilise the negative charge following hydride addition. FADH2 then leaves the active site and is replaced by a new FAD while also regenerating the native state of the enzyme. This step is inferred by the curator.

Step 3. Asp149 deprotonates a saturated bond, allowing FAD to abstract a hydride. Arg152 and Arg148 acidify the saturated bond while Glu31 binds the FAD in place.

Step 4. FADH- accepts a proton to stabilise the negative charge following hydride addition. FADH2 then leaves the active site and is replaced by a new FAD while also regenerating the native state of the enzyme. This step is inferred by the curator.

Step 5. Asp149 removes a proton from a saturated bond, allowing FAD to abstract a hydride. Arg152 and Arg148 acidify the saturated bond while Glu31 binds the FAD in place using electrostatic interactions.

Step 6. FADH- accepts a proton to stabilise the negative charge following hydride addition. FADH2 then leaves the active site and is replaced by a new FAD while also regenerating the native state of the enzyme.

Step 7. Arg152 and Arg148 polarise the C = C double bond, thereby acidifying the neighbouring C-H functionality. Asp149 abstracts a proton from the substrate, facilitating a hydride transfer to FAD from the substrate and forms a C-C double bond.

Step 8. FADH- accepts a proton to stabilise the negative charge following hydride addition. FADH2 then leaves the active site and is replaced by a new FAD while also regenerating the native state of the enzyme. This step is inferred by the curator.

Products.

Contributors

Noa Marson, Marko Babić