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Information on EC 4.2.3.61 - 5-epiaristolochene synthase and Organism(s) Nicotiana tabacum and UniProt Accession Q40577

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EC Tree
     4 Lyases
         4.2 Carbon-oxygen lyases
             4.2.3 Acting on phosphates
                4.2.3.61 5-epiaristolochene synthase
IUBMB Comments
Initial cyclization gives (+)-germacrene A in an enzyme bound form which is not released to the medium.
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This record set is specific for:
Nicotiana tabacum
UNIPROT: Q40577
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The taxonomic range for the selected organisms is: Nicotiana tabacum
The enzyme appears in selected viruses and cellular organisms
Synonyms
eas12, 5-epiaristolochene synthase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
PATHWAY SOURCE
PATHWAYS
-
-
SYSTEMATIC NAME
IUBMB Comments
(2E,6E)-farnesyl-diphosphate diphosphate-lyase [(+)-5-epiaristolochene-forming]
Initial cyclization gives (+)-germacrene A in an enzyme bound form which is not released to the medium.
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
(+)-5-epiaristolochene + diphosphate
show the reaction diagram
(2E,6E)-farnesyl diphosphate
(+)-5-epiaristolochene + diphosphate
show the reaction diagram
(2E,6E)-farnesyl diphosphate
5-epi-aristolochene + diphosphate
show the reaction diagram
-
-
-
?
(2E,6Z)-6-fluorofarnesyl diphosphate
(-)-1-fluorogermacrene A
show the reaction diagram
-
the fluoro substitution at the C6 position of farnesyl diphosphate has negligible effects on enzyme binding, substrate orientation, diphosphate ionization, and the initial 1,10 ring closure catalyzed by TEAS
sole product, 58% yield
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
presence of 2 Mg2+ ions
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0023 - 0.0081
(2E,6E)-farnesyl diphosphate
0.0016 - 0.014
(2E,6E)-farnesyl diphosphate
0.0197
(2E,6Z)-6-fluorofarnesyl diphosphate
-
pH 7.0, 22°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.005 - 0.048
(2E,6E)-farnesyl diphosphate
0.042 - 0.095
(2E,6E)-farnesyl diphosphate
0.11
(2E,6Z)-6-fluorofarnesyl diphosphate
-
pH 7.0, 22°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5 - 6.7
(2E,6E)-farnesyl diphosphate
5.6
(2E,6Z)-6-fluorofarnesyl diphosphate
-
pH 7.0, 22°C
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
wild-type, no activity above
65
thermostable mutant, active up to
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
5EAS_TOBAC
548
0
62987
Swiss-Prot
other Location (Reliability: 2)
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
to 2.2-2.8 A resolution, of TEAS alone and in complexes with two different substrate analogs. TEAS consists entirely of alpha-helices and short connecting loops and turns, and is organized into two structural domains. Two Mg2+ ions are coordinated on opposite sides of the entrance to the active site pocket, and constitute a diphosphate binding site. Asp301 coordinates a Mg2+ in the native TEAS structure, and the side chain carboxyl of Glu379 provides a longer range interaction. Asp305 provides an additional coordination bond in the enzyme with substrate analogs bound. Asp301 and Asp305 are part of a -DDXXD- sequence. Asp301 directly contacts Mg2+, whereas Asp302 demonstrates no direct metal coordination. The side chains of Asp444, Thr448, Glu452, and one water molecule the second coordinate Mg2+. In the native TEAS structure, the A-C and J-K loops and the residues NH2-terminal of residue 36 are disordered
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Y520F
3% of wild-type catalytic efficiency, reaction prooduct is germacrene A
additional information
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38
wild-type, denaturation
83
thermostable mutant, denaturation
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant protein
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
expression in Escherichia coli
-
expression in Petunia hybrida infiltrated with Agrobacterium tumefaciens harboring the enzyme gene
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
method for the recovery of full-length cDNAs from predicted terpene synthase genes containing introns. The approach utilizes Agrobacterium-mediated transient expression coupled with a reverse transcription-polydeoxyribonucleotide chain reaction assay to facilitate expression cloning of processed transcripts. Subsequent expression of intronless cDNAs in a suitable prokaryotic host provides for direct functional testing of the encoded gene product
synthesis
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
O'Maille, P.E.; Chappell, J.; Noel, J.P.
Biosynthetic potential of sesquiterpene synthase: alternative product of tobacco 5-epi-aristolochene synthase
Arch. Biochem. Biophys.
448
73-82
2006
Nicotiana tabacum
Manually annotated by BRENDA team
Wu, S.; Schoenbeck, M.A.; Greenhagen, B.T.; Takahashi, S.; Lee, S.; Coates, R.M.; Chappell, J.
Surrogate splicing for functional analysis of sesquiterpene synthase genes
Plant Physiol.
138
1322-1333
2005
Nicotiana tabacum (Q7X9A3)
Manually annotated by BRENDA team
Faraldos, J.A.; OMaille, P.E.; Dellas, N.; Noel, J.P.; Coates, R.M.
Bisabolyl-derived sesquiterpenes from tobacco 5-epi-aristolochene synthase-catalyzed cyclization of (2Z,6E)-farnesyl diphosphate
J. Am. Chem. Soc.
132
4281-4289
2010
Nicotiana tabacum
Manually annotated by BRENDA team
Back, K.; Yin, S.; Chappell, J.
Expression of a plant sesquiterpene cyclase gene in Escherichia coli
Arch. Biochem. Biophys.
315
527-532
1994
Nicotiana tabacum
Manually annotated by BRENDA team
Brodelius, M.; Lundgren, A.; Mercke, P.; Brodelius, P.
Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum
Eur. J. Biochem.
269
3570-3577
2002
Nicotiana tabacum
Manually annotated by BRENDA team
Rising, K.; Starks, C.; Noel, J.; Chappell, J.
Demonstration of germacrene A as an intermediate in 5-epi-aristolochene synthase catalysis
J. Am. Chem. Soc.
122
1861-1866
2000
Nicotiana tabacum (Q40577)
-
Manually annotated by BRENDA team
Starks, C.; Back, K.; Chappell, J.; Noel, J.
Structural basis for cyclic terpene biosynthesis by tobacco 5-epi-aristolochene synthase
Science
277
1815-1820
1997
Nicotiana tabacum (Q40577)
Manually annotated by BRENDA team
Diaz, J.E.; Lin, C.S.; Kunishiro, K.; Feld, B.K.; Avrantinis, S.K.; Bronson, J.; Greaves, J.; Saven, J.G.; Weiss, G.A.
Computational design and selections for an engineered, thermostable terpene synthase
Protein Sci.
20
1597-1606
2011
Nicotiana tabacum (Q40577)
Manually annotated by BRENDA team