Information on EC 2.5.1.96 - 4,4'-diapophytoene synthase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
2.5.1.96
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RECOMMENDED NAME
GeneOntology No.
4,4'-diapophytoene synthase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2 (2E,6E)-farnesyl diphosphate = 15-cis-4,4'-diapophytoene + 2 diphosphate
show the reaction diagram
2 (2E,6E)-farnesyl diphosphate = diphosphate + presqualene diphosphate
show the reaction diagram
(1a)
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presqualene diphosphate = 15-cis-4,4'-diapophytoene + diphosphate
show the reaction diagram
(1b)
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
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Carotenoid biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
farnesyl-diphosphate:farnesyl-diphosphate farnesyltransferase (4,4'-diapophytoene forming)
Requires Mn2+. Typical of Staphylococcus aureus and some other bacteria such as Heliobacillus sp.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene crtM
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Manually annotated by BRENDA team
gene crtM
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Manually annotated by BRENDA team
gene crtM
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Manually annotated by BRENDA team
gene crtM
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
all of the strains tested contain the genes crtN and crtM arranged as an operon; different strains produce significative amounts of the yellow C30 carotenoid 4,4'-diaponeurosporene in the range 1.8 to 54 mg/kg of dry cell weight
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(2E,6E)-farnesyl diphosphate
15-cis-4,4'-diapophytoene + 2 diphosphate
show the reaction diagram
2 (2E,6E)-farnesyl diphosphate
cis-4,4'-diapophytoene + 2 diphosphate
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
(2E,6E)-farnesyl diphosphate
15-cis-4,4'-diapophytoene + 2 diphosphate
show the reaction diagram
additional information
?
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(3R)-3-biphenyl-4-yl-1-azabicyclo[2.2.2]octan-3-ol
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i.e. BPH-651, forms two crystal structures in complex with the enzyme. In both, the quinuclidine headgroup binds in the allylic S1 site with the side chain in S2, but in the presence of diphosphate and Mg2+, the quinuclidine's cationic center interacts with diphosphate and three Mg2+, mimicking a transition state involved in diphosphate ionization
(S)-1-phosphono-4-(3-phenoxyphenyl)butylsulfonic acid
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far more active than (R)-compound both in vitro an in cells
1-phosphono-4-[3-(3,4-difluorophenoxy)phenyl]butylsulfonic acid
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1-phosphono-4-[3-(3-fluorophenoxy)phenyl]butylsulfonic acid
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1-phosphono-4-[3-(4-fluorophenoxy)phenyl]butylsulfonic acid
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1-phosphono-4-[3-(4-propylphenoxy)phenyl]butylsulfonic acid
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potent inhibitor in cell-based assay
2-(4-phenoxyphenoxy)ethyl thiocyanate
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i.e. WC-9, binds to the S2 site with its -SCN group surrounded by four hydrogen bond donors
4-(3-phenoxyphenyl)-1-phosphonobutane-1-sulfonic acid
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i.e. BPH-652
4-(4'-butylbiphenyl-4-yl)-1-phosphonobutane-1-sulfonic acid
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i.e. BPH-698
4-(4-biphenyl)butyldiphosphonic acid
4-(biphenyl-4-yl)-1-phosphonobutane-1-sulfonic acid
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i.e. BPH-700
4-[4-(4-trifluoromethylphenyl)phenyl]butyldiphosphonic acid
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lapaquistat acetate
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docking analysis, interaction with residues H18, R45, D48, D52, Y129, Q165, N168 and D172
N-[(2E)-3,7-dimethylocta-2,6-dien-1-yl]-N'-[(1R,3S,5R,7R)-tricyclo[3.3.1.1-3,7-]dec-2-yl]ethane-1,2-diamine
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i.e. SQ-109, forms two crystal structurs in complex with the enzyme. In one, the geranyl side chain binds to either S1 or S2 and the adamantane headgroup binds to S1. In the second, the side chain binds to S2 while the headgroup binds to S1
squalestatin
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docking analysis, interaction with residues H18, R45, D48, D52, Y129, Q165, N168 and D172
zaragozic acid A
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crystallization data of complex
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0012
(2E,6E)-farnesyl diphosphate
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pH 7.7, 37C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0000015
4-(3-phenoxyphenyl)-1-phosphonobutane-1-sulfonic acid
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pH not specified in the publication, temperature not specified in the publication
0.000135
4-(4'-butylbiphenyl-4-yl)-1-phosphonobutane-1-sulfonic acid
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pH not specified in the publication, temperature not specified in the publication
0.0000002
4-(4-biphenyl)butyldiphosphonic acid
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pH not specified in the publication, temperature not specified in the publication
0.000006
4-(biphenyl-4-yl)-1-phosphonobutane-1-sulfonic acid
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pH not specified in the publication, temperature not specified in the publication
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0014
(S)-1-phosphono-4-(3-phenoxyphenyl)butylsulfonic acid
Staphylococcus aureus
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pH 7.4, 20C
0.0022
1-phosphono-4-[3-(3,4-difluorophenoxy)phenyl]butylsulfonic acid
Staphylococcus aureus
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pH 7.4, 20C
0.0021
1-phosphono-4-[3-(3-fluorophenoxy)phenyl]butylsulfonic acid
Staphylococcus aureus
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pH 7.4, 20C
0.0023
1-phosphono-4-[3-(4-fluorophenoxy)phenyl]butylsulfonic acid
Staphylococcus aureus
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pH 7.4, 20C
0.0022
1-phosphono-4-[3-(4-propylphenoxy)phenyl]butylsulfonic acid
Staphylococcus aureus
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pH 7.4, 20C
0.0005
4-(4-biphenyl)butyldiphosphonic acid
Staphylococcus aureus
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pH 7.4, 20C
0.0002
4-[4-(4-trifluoromethylphenyl)phenyl]butyldiphosphonic acid
Staphylococcus aureus
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pH 7.4, 20C
PDB
SCOP
CATH
ORGANISM
UNIPROT
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30121
x * 30121, calculated
33419
x * 33419, calculated
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
docking analysis of lapaquistat acetate and squalestatin to crtM. Residues H18, R45, D48, D52, Y129, Q165, N168 and D172 interact with the inhibitors
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in complex with three inhibitors bound, to about 2.1 A resolution. with inhibitor (3R)-3-biphenyl-4-yl-1-azabicyclo[2.2.2]octan-3-ol, enzyme forms two crystal structures. In both, the quinuclidine headgroup binds in the allylic S1 site with the side chain in S2, but in the presence of diphosphate and Mg2+, the quinuclidine's cationic center interacts with diphosphate and three Mg2+, mimicking a transition state involved in diphosphate ionization. Inhibitor 2-(4-phenoxyphenoxy)ethyl thiocyanate binds to the S2 site with its -SCN group surrounded by four hydrogen bond donors. Inhibitor N-[(2E)-3,7-dimethylocta-2,6-dien-1-yl]-N'-[(1R,3S,5R,7R)-tricyclo[3.3.1.1-3,7-]dec-2-yl]ethane-1,2-diamine forms two crystal structurs in complex with the enzyme. In one, the geranyl side chain binds to either S1 or S2 and the adamantane headgroup binds to S1. In the second, the side chain binds to S2 while the headgroup binds to S1
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in with its reaction intermediate, presqualene diphosphate, the dehydrosqualene product, as well as a series of inhibitors. The results indicate that, on initial diphosphate loss, the primary carbocation so formed bends down into the interior of the protein to react with C2,3 double bond in the prenyl acceptor to form presqualene diphosphate, with the lower two-thirds of both presqualene diphosphate chains occupying essentially the same positions as found in the two farnesyl chains in the substrates. The second-half reaction is then initiated by the presqualene diphosphate returning back to the Mg2+ cluster for ionization, with the resultant dehydrosqualene so formed being trapped in a surface pocket
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mutant Y248A in complex with zaragozic acid A, to 2.1 A resolution. Crystals grow in the hexagonal space group P3121 and contain two molecules per asymmetric unit. The active site of each protein is occupied by a molecule zazgozic acid A. The highly oxygenated core structure contacts residues 19SKSF22. The C-1 lipophilic tail extends into the narrow pocket which is lined with hydrophobic residues that help to stabilize the interaction with the isoprenoid moiety of the donor farnesyl diphosphate, S1 site.The side chains of Phe22 and Phe26 are moved toward the bottom of the active site, and the orientation of the Tyr41 side chain provides sufficient space for stabilization of the zaragozic acid A C-1 unit in the S1 site
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native protein to 1.58 A resolution. CrtM crystallizes in the P3221 space group and there is one molecule per asymmetric unit. The overall fold shows similarity to that seen in human squalene synthase. In the complex with substrate analogue farnesyl thiodiphosphate, two farnesyl thiodiphosphate molecules are found in the large central cavity. Their diphosphate head groups interact with three Mg2+ ions, which in turn interact with Asp residues in two conserved Asp-X-X-X-Asp repeats. The space group of the complex is P3121, and there are two molecules per asymmetric unit. In docking studies with phosphonosulfonate inhibitors, only one phosphonosulfonate is bound per CrtM. All three inhibitors tested have different binding modes. 4-(3-phenoxyphenyl)-1-phosphonobutane-1-sulfonic acid binds into the farnesyl thiodiphosphate-1 site with two Mg2+, 4-(4'-butylbiphenyl-4-yl)-1-phosphonobutane-1-sulfonic acid binds into the farnesyl thiodiphosphate-2 site with only one Mg2+, and 4-(biphenyl-4-yl)-1-phosphonobutane-1-sulfonic acid binds into the farnesyl thiodiphosphate-2 site with no Mg2+. The phosphonosulfonate side chains do closely track the locations of the two farnesyl thiodiphosphate inhibitor side chains
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant protein
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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gene crtM, DNA and amino acid sequence determination and analysis, genetic organization, sequence comparisons, phylogenetic tree, functional pathway complementation by recombinant enzyme expression in Escherichia coli
gene ysqs, recombinant expression of the engineered mutant enzyme in Escherichia coli strain XL1-Blue, coexpression with gene crtN encoding DSQ desaturase from Staphylococcus aureus in Escherichia coli results in carotinoid production and accumulation of C30 carotenoid pigments, which does not happen with coexpression of gene crtI encoding Pantoea ananatis phytoene desaturase, carotenoid pigment analysis, overview
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recombinant expression of the endgineered enzymes in Escherichia coli strain XL1-Blue, coexpression with gene crtN encoding DSQ desaturase from Staphylococcus aureus in Escherichia coli results in carotinoid production and accumulation of C30 carotenoid pigments, which does not happen with coexpression of gene crtI encoding Pantoea ananatis phytoene desaturase, carotenoid pigment analysis, overview
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recombinant expression of the engineered mutant enzyme in Escherichia coli strain XL1-Blue, coexpression with gene crtN encoding DSQ desaturase from Staphylococcus aureus in Escherichia coli results in carotinoid production and accumulation of C30 carotenoid pigments, which does not happen with coexpression of gene crtI encoding Pantoea ananatis phytoene desaturase, carotenoid pigment analysis, overview
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Q33R/D34N/S38N
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directed evolution of human Q33R/D34N/S38N mutant squalene synthase, EC 2.5.1.21, in an attempt to mimic the activity of dehydroqsqualene synthase, EC 2.5.1.96, by replacement of hsqs in pAC-hsqs with the Saccharomyces mutant gene ysqs G856A/G1125A/T1128C/T1176C
G856A/G1125A/T1128C/T1176C
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directed evolution of squalene synthase, EC 2.5.1.21, in an attempt to mimic the activity of dehydroqsqualene synthase, EC 2.5.1.96, by mutation of gene ysqs
A726G, A850G
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mutant with phytoene synthase activity, decrease in dehydrosqualene synthase activity
F26L
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mutation is sufficient to gain phytoene synthase activity, decrease in dehydrosqualene synthase activity
F26L/E149G
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mutant with phytoene synthase activity, decrease in dehydrosqualene synthase activity
F26L/F267S
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mutant with phytoene synthase activity, decrease in dehydrosqualene synthase activity
F26L/V43M
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mutant with phytoene synthase activity, decrease in dehydrosqualene synthase activity
F26S/I40T
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mutant with phytoene synthase activity, decrease in dehydrosqualene synthase activity
H12R/F26L/D27G/90G/K97R/H207R
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mutant with phytoene synthase activity, decrease in dehydrosqualene synthase activity
K20E/F26S
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mutant with phytoene synthase activity, decrease in dehydrosqualene synthase activity
M4V/H12R/F59S/Q81R/E180G
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mutant with phytoene synthase activity, without decrease in dehydrosqualene synthase activity
Y129A
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no activity with substrate farnesyl diphophate, 7% residual activity with geranyl diphosphate
Y248A
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crystallization data of complex with zaragozic acid A
F26L
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mutation is sufficient to gain phytoene synthase activity, decrease in dehydrosqualene synthase activity
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F26L/E149G
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mutant with phytoene synthase activity, decrease in dehydrosqualene synthase activity
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F26L/V43M
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mutant with phytoene synthase activity, decrease in dehydrosqualene synthase activity
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K20E/F26S
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mutant with phytoene synthase activity, decrease in dehydrosqualene synthase activity
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additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
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construction of an in vitro C30 carotenoid pathway based on recombinant Escherichia coli farnesyl diphosphate synthase and 4,4'-diapophytoene synthase. The 4,4'-diapophytoene product is unstabel in aqueous buffer. In situ extraction using an aqueous-organic two-phase system results in a 100% conversion of isopentenyl diphosphate and dimethylallyl diphosphate into 4,4'-diapophytoene synthase
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