Information on EC 2.5.1.82 - hexaprenyl diphosphate synthase [geranylgeranyl-diphosphate specific]

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The expected taxonomic range for this enzyme is: Sulfolobus solfataricus

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EC NUMBER
COMMENTARY hide
2.5.1.82
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RECOMMENDED NAME
GeneOntology No.
hexaprenyl diphosphate synthase [geranylgeranyl-diphosphate specific]
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REACTION
REACTION DIAGRAM
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ORGANISM
UNIPROT
LITERATURE
geranylgeranyl diphosphate + 2 isopentenyl diphosphate = 2 diphosphate + all-trans-hexaprenyl diphosphate
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
isoprenoid biosynthesis
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Terpenoid backbone biosynthesis
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Biosynthesis of secondary metabolites
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SYSTEMATIC NAME
IUBMB Comments
geranylgeranyl-diphosphate:isopentenyl-diphosphate transferase (adding 2 isopentenyl units)
The enzyme prefers geranylgeranyl diphosphate to farnesyl diphosphate as an allylic substrate and does not show activity for geranyl diphosphate and dimethylallyl diphosphate. Requires Mg2+ [1].
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CAS REGISTRY NUMBER
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83745-07-7
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
farnesyl diphosphate + 3 isopentenyl diphosphate
3 diphosphate + all-trans-hexaprenyl diphosphate
show the reaction diagram
geranyl diphosphate + 4 isopentenyl diphosphate
4 diphosphate + all-trans-hexaprenyl diphosphate
show the reaction diagram
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?
geranylgeranyl diphosphate + 2 isopentenyl diphosphate
2 diphosphate + all-trans-hexaprenyl diphosphate
show the reaction diagram
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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
geranylgeranyl diphosphate + 2 isopentenyl diphosphate
2 diphosphate + all-trans-hexaprenyl diphosphate
show the reaction diagram
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
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the enzyme does not show activity without a divalent cation and prefers Mg2+ to other cations such as Mn2+ and Ca2+. At concentrations ranging from 2.5 to 50 mM, the effect of Mg2+ does not change greatly
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Triton X-100
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approximately increases the activity of the enzyme 2fold and retains the effect at a similar level through various concentrations from 0.01 to 1%
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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PDB
SCOP
CATH
ORGANISM
UNIPROT
Sulfolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)
Sulfolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
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LITERATURE
32000
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x * 32000, SDS-PAGE
32274
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x * 32274, calculated from sequence
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SUBUNITS
ORGANISM
UNIPROT
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LITERATURE
homodimer
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
D81C and W136E mutant crystals are grown at room temperature by the hanging drop vapor diffusion method from a reservoir solution containing 10% (w/v) polyethyleneglycol 8000, 2.4 M lithium chloride, and 100 mM Tris (pH 8.0). Crystal structure of the homodimeric C30-HexPPs resembles those of other trans-prenyltransferases, including farnesyl pyrophosphate synthase (FPPs) and octaprenyl pyrophosphate synthase
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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expression in Escherichia coli BL21(DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D81C
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crystal structure of mutant enzyme
W136E
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crystal structure of mutant enzyme
D81C
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crystal structure of mutant enzyme
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W136E
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crystal structure of mutant enzyme
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additional information
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introduction of mutations mimicking the chain-length determination region of short-chain enzymes into Sulfolobus solfataricus hexaprenyldiphosphate synthase and characterization of the mutated enzymes. To mimic short-chain enzymes without geranylgeranyl diphosphate synthase type III, the typical amino acid residues observed in the region of enzymes of each group are introduced into Sulfolobus solfataricus hexaprenyldiphosphate synthase. Several successive amino acids before the first aspartate rich motif of Sulfolobus solfataricus hexaprenyldiphosphate synthase are replaced with those of geranylgeranyl diphosphate synthase from Saccharomyces cerevisiae to construct a mutant mimicking geranylgeranyl diphosphate synthase type III. To facilitate purification, (His)6-tag is introduced at the N-terminal of each enzyme. Introduction of the tag does not significantly affect the activity and product specificity of wild-type Sulfolobus solfataricus hexaprenyldiphosphate synthase. The mutant HPS-F1, HPS-GG1, and HPS-GG3, mimicking the chain-length determination regions of eukaryotic farnesyl diphosphate synthases, archaeal geranylgeranyl diphosphate synthases, and eukaryotic geranylgeranyl diphosphate synthases, respectively, sustain high activity comparable to that of the wild type enzyme, except for the facts that HPS-F1 shows reduced activity when (all-trans)-geranylgeranyl diphosphate is used as the allylic substrate (5.5% of the activity of wild-type HPS) and that the activity of HPS-GG3 toward geranyl diphosphate is relatively low (16% of that of wild type hexaprenyldiphosphate synthase). HPS-GG2, possessing the insertion sequence to mimic the chain-length determination region of geranylgeranyl diphosphate synthase type II, significantly decreases enzyme activity toward each allylic substrate (0.8%, 8.3%, and 45% of the activities of wild-type HPS for geranyl diphosphate, farnesyl diphosphate, and (all-trans)-geranylgeranyl diphosphate, respectively). HPS-F2 mimicking the chain-length determination region of farnesyl diphosphate synthase type II, which has both the insertion and the additional mutation before first aspartate rich motif, shows no activity, although the same amounts of enzymes are used. The inactivation of HPS-F2 is considered to arise from either the difference of its insertion sequence from that of HPS-GG2 or the introduction of bulky amino acid before first aspartate rich motif. When geranyldiphosphate and farnesyl diphosphate are used as the allylic substrate, the main product of HPS-F1 is geranylgeranyl diphosphate. A slight amount of C25 product is produced when (all-trans)-geranylgeranyl diphosphate is the substrate. These results and the low reactivity of HPS-F1 with (all-trans)-geranylgeranyl diphosphate indicate that the product specificity of the mutant has been changed into that of geranylgeranyl diphosphate synthase. The conversion of Sulfolobus solfataricus hexaprenyldiphosphate synthase into geranylgeranyl diphosphate synthase, not into farnesyl diphosphate synthase, by the mutation mimicking the chain-length determination region of farnesyl diphosphate synthase type I might reflect the structural difference between hexaprenyldiphosphate synthase and type I farnesyl diphosphate synthases existing at positions other than the mutated sites. HPS-GG1 maily produces hexaprenyldiphosphate, just as wild-type hexaprenyl diphosphate synthase does. It also yields small amounts of C35 product and, when geranyl diphosphate is used as the substrate, the C40 product. Either the conformation of the chain-length determination region or the orientation of the carbon-chain of products in the cavity of the enzyme is considerably different between the archaeal geranylgeranyl diphosphate synthase and hexaprenyl diphosphate synthase
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LINKS TO OTHER DATABASES (specific for EC-Number 2.5.1.82)
ExplorEnz
ExPASy
KEGG
MetaCyc
SABIO-RK
NCBI: PubMed, Protein, Nucleotide, Structure, Genome, OMIM
IUBMB Enzyme Nomenclature
PROSITE Database of protein families and domains
InterPro (database of protein families, domains and functional sites)