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Information on EC 4.2.3.9 - aristolochene synthase and Organism(s) Aspergillus terreus and UniProt Accession Q9UR08

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EC Tree
     4 Lyases
         4.2 Carbon-oxygen lyases
             4.2.3 Acting on phosphates
                4.2.3.9 aristolochene synthase
IUBMB Comments
The initial internal cyclization produces the monocyclic intermediate germacrene A; further cyclization and methyl transfer converts the intermediate into aristolochene. While in some species germacrene A remains as an enzyme-bound intermediate, it has been shown to be a minor product of the reaction in Penicillium roqueforti (see also EC 4.2.3.23, germacrene-A synthase). The enzyme from Penicillium roqueforti requires Mg2+. Mn2+ can partially substitute, at low concentrations. Aristolochene is the likely parent compound for a number of sesquiterpenes produced by filamentous fungi.
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Aspergillus terreus
UNIPROT: Q9UR08
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The taxonomic range for the selected organisms is: Aspergillus terreus
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
Synonyms
sesquiterpene cyclase, aristolochene synthase, 5-epi-aristolochene synthase, farnesylpyrophosphate cyclase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
aristolochene synthase
5-epi-aristolochene synthase
-
-
aristolochene synthase
-
sesquiterpene synthase, class I enzyme
AS
-
-
-
-
cyclase, farnesyl pyrophosphate
-
-
-
-
farnesylpyrophosphate cyclase
-
-
-
-
FPP-carbocyclase
-
-
-
-
sesquiterpene cyclase
-
-
-
-
synthase, aristolochene
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
cyclization
cyclization
-
-
internal cyclization
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
(2E,6E)-farnesyl diphosphate diphosphate-lyase (cyclizing, aristolochene-forming)
The initial internal cyclization produces the monocyclic intermediate germacrene A; further cyclization and methyl transfer converts the intermediate into aristolochene. While in some species germacrene A remains as an enzyme-bound intermediate, it has been shown to be a minor product of the reaction in Penicillium roqueforti [5] (see also EC 4.2.3.23, germacrene-A synthase). The enzyme from Penicillium roqueforti requires Mg2+. Mn2+ can partially substitute, at low concentrations. Aristolochene is the likely parent compound for a number of sesquiterpenes produced by filamentous fungi.
CAS REGISTRY NUMBER
COMMENTARY hide
94185-89-4
-
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
aristolochene + diphosphate
show the reaction diagram
-
-
-
?
2-fluorofarnesyl diphosphate
2-fluorogermacrene A + diphosphate
show the reaction diagram
two products are identified in a 95/5 ratio by GS-MS
-
-
?
2-trans,6-trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
-
-
?
farnesyl diphosphate
(+)-aristolochene + diphosphate
show the reaction diagram
The universal sesquiterpene precursor farnesyl diphosphate (15-carbon isoprenoid) is cyclized in an Mg2-dependent reaction to form the bicyclic hydrocarbon aristolochene and a diphosphate anion coproduct
-
-
?
2-fluorofarnesyl diphosphate
2-fluorogermacrene A + diphosphate
show the reaction diagram
-
-
-
-
?
2-trans,6-trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
terpenoid biosynthesis
-
-
r
trans,trans-farnesyl diphosphate
(+)-aristolochene + diphosphate
show the reaction diagram
-
-
(+)-aristolochene is the only product
-
?
trans,trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
additional information
?
-
no activity with farnesyl S-thiolodiphosphate
-
-
?
NATURAL SUBSTRATE
NATURAL 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
aristolochene + diphosphate
show the reaction diagram
-
-
-
?
2-trans,6-trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
-
-
?
farnesyl diphosphate
(+)-aristolochene + diphosphate
show the reaction diagram
The universal sesquiterpene precursor farnesyl diphosphate (15-carbon isoprenoid) is cyclized in an Mg2-dependent reaction to form the bicyclic hydrocarbon aristolochene and a diphosphate anion coproduct
-
-
?
2-trans,6-trans-farnesyl diphosphate
aristolochene + diphosphate
show the reaction diagram
-
terpenoid biosynthesis
-
-
r
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
-
can substitute for Mg2+ over the concentration range of 0.16 -5.0 mM
Mn2+
-
Mn2+ can replace Mg2+ at 0.1-0.2 mM
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
12,13-difluorofarnesyl diphosphate
incubation of 12,13-difluorofarnesyl diphosphate with for 30 h does not generate any pentane-extractable products based on GC-MS analysis
Mn2+
-
above 1.0 mM
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000042 - 0.00041
(2E,6E)-farnesyl diphosphate
0.0000135 - 0.0039
trans,trans-farnesyl diphosphate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00041 - 0.045
(2E,6E)-farnesyl diphosphate
0.000076 - 0.0173
trans,trans-farnesyl diphosphate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.1 - 370
(2E,6E)-farnesyl diphosphate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0236
-
native enzyme
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
-
recombinant enzyme, HEPES buffer
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
ARIS_ASPTE
320
0
36481
Swiss-Prot
other Location (Reliability: 3)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70000
dimer, native gel analysis
36480
-
x * 36480, calculation from nucleotide sequence
39000
-
x * 39000, SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
tetramer
crystal structure
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystallization by the hanging drop, vapor diffusion method at 4°C, the crystal structure determined from crystals soaked with farnesyl diphosphate reveals the binding of intact farnesyl diphosphate to monomers A-C, and the binding of diphosphate anion and Mg2+ to monomer D. The structure of the complex with 2-fluorofarnesyl diphosphate reveals 2-fluorofarnesyl diphosphate binding to all subunits of the tetramer, with Mg2+B accompanying the binding of this analogue only in monomer D. The structure of the complex with 12,13-difluorofarnesyl diphosphate reveals the binding of intact 12,13-difluorofarnesyl diphosphate to monomers A-C in the open conformation and the binding of diphosphate anion, Mg2+B, and Mg2+C to monomer D in a predominantly closed conformation
molecular dynamics simulations based on structure PDB entry 2OA6. The substrate farnesyl diphosphate binds first, followed by three magnesium ions in sequence, and, after reaction, the release of aristolochene and two magnesium ions followed by the final magnesium ion and diphosphate. Binding of farnesyl diphosphate leads to an increased level of sampling of open conformations, allowing the first two magnesium ions to bind. The closed enzyme conformation is maintained with a diphosphate moiety and two magnesium ions bound. The open-to-closed transition reduces flexibility around the active site entrance, partly through a lid closing over it
the structure of recombinant aristolochene synthase at a resolution of 2.2 A and its complex with diphosphate and three Mg2+ ions at 2.15 A is reported
wild type and mutant enzymes in complex with (2E,6E)-farnesyl diphosphate or 4, sitting drop vapor diffusion method, using 20% PEG 3350 as precipitant
X-ray crystal structure of aristolochene synthase complexed with three Mg2+ ions and the unreactive substrate analogue farnesyl-S-thiolodiphosphate, showing that the substrate diphosphate group is anchored by metal coordination and hydrogen bond interactions. The binding conformation of farnesyl-S-thiolodiphosphate directly mimics that expected for productively bound farnesyl diphosphate, with the exception of the precise alignment of the C-S bond with regard to the C10-C11 pi system that would be required for C1-C10 bond formation in the first step of catalysis. Crystal structures of aristolochene synthase complexed with Mg2+3-diphosphate and ammonium or iminium analogues of bicyclic carbocation intermediates proposed for the natural cyclization cascade show various binding orientations for these bicyclic analogues, which appear to be driven by favorable electrostatic interactions between the positively charged ammonium group of the analogue and the negatively charged diphosphate anion. The active site is sufficiently flexible to accommodate analogues with partially or completely incorrect stereochemistry
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
N299A
the mutation has decreasing effects on catalysis but significant effects on sesquiterpene product distributions of 50% germacrene A and 50% aristolochene
N299A/S303A
the decreasing have modest effects on catalysis but significant effects on sesquiterpene product distributions with germacrene A as main product and (E)-nerolidol and aristolochene as by-products
N299L
the mutation has decreasing effects on sesquiterpene product distributions with germacrene A and aristolochene as main product and (E)-nerolidol as by-product
Q151E
the mutation has decreasing effects on catalysis but significant effects on sesquiterpene product distributions with aristolochene and germacrene A as main products, and (E)-nerolidol and (E,E)-farnesol as by-products
Q151H
the mutation has decreasing effects on catalysis but significant effects on sesquiterpene product distributions with germacrene A and (E,E)-farnesol as main products and (E)-nerolidol and aristolochene as by-products
S303A
the mutation has increasing effects on catalysis and significant effects on sesquiterpene product distributions with germacrene A and (E)-nerolidol as by-products
S303D
the mutation has decreasing effects on catalysis but significant effects on sesquiterpene product distributions with germacrene A and (E,E)-farnesol as main products and (E)-nerolidol and aristolochene as by-products
S303H
the mutation has decreasing effects on catalysis but significant effects on sesquiterpene product distributions with germacrene A as main product and (E,E)-farnesol, (E)-nerolidol and aristolochene as by-products
E227D
-
kcat/KM is 1182fold higher than wild-type value, in contrast to wild-type enzyme that exclusively produces aristolochene from trans,trans-farnesyl diphosphate, the mutant enzyme produces 26% aristolochene and 74% germacrene A
E227Q
-
inactive mutant enzyme
N219D
-
kcat/KM is 6667fold higher than wild-type value, in contrast to wild-type enzyme that exclusively produces aristolochene from trans,trans-farnesyl diphosphate, the mutant enzyme produces 44% aristolochene and 56% germacrene A
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
Ni-NTA column chromatography, HiTrap Q column chromatography, and Superdex 26/60 gel filtration
using DE52 anion exchange resin, a methyl HIC, a Sephadex G-25, and a High Q column
native and recombinant enzyme
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
into the pET11 vector for expression in Escherichia coli BL21DE3pLysS cells
recombinant aristolochene synthase is expressed in Escherichia coli BL21(DE3)pLysS
high-level expression in Escherichia coli
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Cane, D.E.; Prabhakaran, P.C.; Oliver, J.S.; McIlwaine, D.B.
Aristolochene biosynthesis. Stereochemistry of the deprotonation steps in the enzymatic cyclization of farnesyl pyrophosphate
J. Am. Chem. Soc.
112
3209-3210
1990
Aspergillus terreus
-
Manually annotated by BRENDA team
Cane, D.E.; Prabhakaran, P.C.; Salaski, E.J.; Harrison, P.M.H.; Noguchi, H.; Rawlings, B.J.
Aristolochene biosynthesis and enzymatic cyclization of farnesyl pyrophosphate
J. Am. Chem. Soc.
111
8914-8916
1989
Aspergillus terreus
-
Manually annotated by BRENDA team
Cane, D.E.; Kang, I.
Aristolochene synthase: purification, molecular cloning, high-level expression in Escherichia coli, and characterization of the Aspergillus terreus cyclase
Arch. Biochem. Biophys.
376
354-364
2000
Aspergillus terreus, Penicillium roqueforti
Manually annotated by BRENDA team
Felicetti, B.; Cane, D.E.
Aristolochene synthase: mechanistic analysis of active site residues by site-directed mutagenesis
J. Am. Chem. Soc.
126
7212-7221
2004
Aspergillus terreus, Penicillium roqueforti (Q03471), Penicillium roqueforti
Manually annotated by BRENDA team
Shishova, E.Y.; Di Costanzo, L.; Cane, D.E.; Christianson, D.W.
X-ray crystal structure of aristolochene synthase from Aspergillus terreus and evolution of templates for the cyclization of farnesyl diphosphate
Biochemistry
46
1941-1951
2007
Aspergillus terreus (Q9UR08), Aspergillus terreus
Manually annotated by BRENDA team
Christianson, D.W.
Unearthing the roots of the terpenome
Curr. Opin. Chem. Biol.
12
141-150
2008
Aspergillus terreus
Manually annotated by BRENDA team
Shishova, E.Y.; Yu, F.; Miller, D.J.; Faraldos, J.A.; Zhao, Y.; Coates, R.M.; Allemann, R.K.; Cane, D.E.; Christianson, D.W.
X-ray crystallographic studies of substrate binding to aristolochene synthase suggest a metal ion binding sequence for catalysis
J. Biol. Chem.
283
15431-15439
2008
Aspergillus terreus (Q9UR08), Aspergillus terreus
Manually annotated by BRENDA team
Chen, M.; Al-lami, N.; Janvier, M.; DAntonio, E.L.; Faraldos, J.A.; Cane, D.E.; Allemann, R.K.; Christianson, D.W.
Mechanistic insights from the binding of substrate and carbocation intermediate analogues to aristolochene synthase
Biochemistry
52
5441-5453
2013
Aspergillus terreus (Q9UR08), Aspergillus terreus
Manually annotated by BRENDA team
van der Kamp, M.W.; Sirirak, J.; ?urek, J.; Allemann, R.K.; Mulholland, A.J.
Conformational change and ligand binding in the aristolochene synthase catalytic cycle
Biochemistry
52
8094-8105
2013
Aspergillus terreus (Q9UR08)
Manually annotated by BRENDA team
Chen, M.; Chou, W.K.; Al-Lami, N.; Faraldos, J.A.; Allemann, R.K.; Cane, D.E.; Christianson, D.W.
Probing the role of active site water in the sesquiterpene cyclization reaction catalyzed by aristolochene synthase
Biochemistry
55
2864-2874
2016
Aspergillus terreus (Q9UR08)
Manually annotated by BRENDA team