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Information on EC 4.2.1.129 - squalene-hopanol cyclase and Organism(s) Alicyclobacillus acidocaldarius and UniProt Accession P33247
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4.2.1.129 squalene-hopanol cyclaseIUBMB Comments
The enzyme produces the cyclization products hopene (cf. EC 5.4.99.17) and hopanol from squalene at a constant ratio of 5:1.
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The taxonomic range for the selected organisms is: Alicyclobacillus acidocaldarius
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Synonyms
SHC, squalene-hopene cyclase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hopan-22-ol = squalene + H2O
overall mechanism of the polycyclization reaction of SHCs and structures of squalene cyclization products, overview
PATHWAY SOURCE
PATHWAYS
-
-, -
SYSTEMATIC NAME
IUBMB Comments
hopan-22-ol hydro-lyase
The enzyme produces the cyclization products hopene (cf. EC 5.4.99.17) and hopanol from squalene at a constant ratio of 5:1.
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
hopan-22-ol
squalene + H2O
-
squalenehopene cyclase from prokaryotes catalyses the conversion of the acyclic molecule of squalene into the pentacyclic triterpenes of hopene and hopanol in the the ratio 5:1. The polycyclization of squalene to hopene consists of sequential ring-forming reaction steps. The polycyclization cascade is initiated by the electrophilic attack of the acidic proton, donated by the DXDD motif, to one of the two terminal double bonds. The polycyclization reaction is quenched by proton elimination from the alternative terminal methyl group of squalene. Based on the X-ray analysis of Alicyclobacilus acidocaldarius, the catalytic base responsible for the deprotonation reaction has been suggested to be a water molecule (named a front water), the polarization of which is enhanced by other waters (named back waters) that construct the hydrogen-bonding network by a combination of seven residues T41, E45, E93, R127, Q262, W133 and Y267. The front water thus polarized can store the proton generated from either Me-29 or Me-30 of hopanyl cation to form hopene, but hopanol is produced if the front water adds as hydroxyl to the C-22 cation of the A-ring instead of accepting the proton
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-
?
additional information
?
-
substrate specificity, overview
-
-
?
additional information
?
-
product pattern of alternative substrates, overview
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
additional information
?
-
product pattern of alternative substrates, overview
-
-
?
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
SHC in vivo is a membrane-associated protein and can be solubilized from cell extracts by nonionic detergents, such as Triton X-100 or octylthioglucopyranoside. The enzyme is attached to the inner side of the cytoplasmic membrane by interactions of hydrophobic residues with the phospholipids. The membrane-binding part of the enzyme is a nonpolar region that is encircled by positive-charged amino acids enforcing the anchoring of the enzyme to the negatively charged surface of the phospholipid membrane
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
enzyme distribution in the different taxa, overview
metabolism
the enzyme converts squalene to hopanol, EC 4.2.1.129, and to hopene, EC 5.4.99.17, but not to tetrahymanol, EC 4.2.1.123, pathway overview
additional information
structure-function relationships of SHCs, active site structure, overview. A protruding part in the center of the nonpolar region contains a lipophilic channel and directs the substrate to the active-site cavity inside the protein. The channel and cavity are separated by a narrow constriction buildup of four amino acids, D376, F166, C435, and F434, that appear to block access to the active site. Residues C435 and F434 are part of a loop that seems to be flexible enough to permit passage of the substrate and the product
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
each subunit consists of alpha-helical domains that build up a dumbbell-shaped structure. The first domain consists of a regular (alpha/alpha)6 barrel structure, whereas the second domain shows an alpha-barrel structure in a less periodic manner
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C435S/D374I/D374V/H451F
site-directed mutagenesis, inactive mutant
D377C/D377N/Y612A
site-directed mutagenesis, the mutant shows an altered product pattern compared to the wild-type enzyme, overview
D377E/D376Q/D376R/D377R/E45K/W406V/W417A/D377C
site-directed mutagenesis, inactive mutant
F365A
site-directed mutagenesis, the mutant shows an altered product pattern compared to the wild-type enzyme, overview
F601A
site-directed mutagenesis, the mutant shows an altered product pattern compared to the wild-type enzyme, overview
F605A
site-directed mutagenesis, the mutant shows an altered product pattern compared to the wild-type enzyme, overview
I261A
site-directed mutagenesis, the mutant shows an altered product pattern compared to the wild-type enzyme, overview
Q262G/Q262A/P263G/P263A
site-directed mutagenesis, the mutant shows an altered product pattern compared to the wild-type enzyme, overview
V381A/D376C
site-directed mutagenesis, inactive mutant
W169F/W169H/W489A/F605K
site-directed mutagenesis, the mutant shows an altered product pattern compared to the wild-type enzyme, overview
Y420A
site-directed mutagenesis, the mutant shows an altered product pattern compared to the wild-type enzyme, overview
Y606A/W23V/W495V/W522V/W533A/W591L/W78S/E35Q/E197Q/D530N/T378A
site-directed mutagenesis, the mutant shows the same product pattern and activity as the wild-type
Y609A/Y612A/L607K
site-directed mutagenesis, the mutant shows an altered product pattern compared to the wild-type enzyme, overview
Y609F
Y612F/D376E/D376G/D377E/D377G/D377Q/E45A/E45D/F365W/T41A/E93A/R127Q/W133A/Y267A/F434A/F437A/W258L/D350N/D421N/D442N/H451R/D447N/D377N/D313N/E535Q/D374E
site-directed mutagenesis, the mutant shows the same product pattern as the wild-type with less enzyme activity
E45A
-
production of hop-22(29)-ene is less throughout the entire temperature range than that by the wild-type. Hop-21(22)ene is not produced
E93A
-
production of hop-22(29)-ene is less throughout the entire temperature range than that by the wild-type. Hop-21(22)ene is not produced
F434A
-
production of hop-22(29)-ene is decreased, production of hopanol is markedly increased at lower temperatures
F437A
-
production of hop-22(29)-ene is decreased, production of hopanol is markedly increased at lower temperatures
G262A
-
the mutant produces hopanol as the main product instead of hop-22(29)-ene. The mutant also produces hop-21(22)ene
P263A
P263G
Q262A
-
the mutation results in a greatly enhanced production of hopanol along with the decreased formation of hopene. A high production of hopanol would be explained as follows. The point mutations could give rise to the perturbation around the front water. This disordered front water cannot correctly act as the catalytic base for the deprotonation reaction to form hopene, and in turn could be placed near to the final hopanyl cation, leading to a high production of hopanol without forming hopene
Q262G
R127Q
-
production of hop-22(29)-ene is less throughout the entire temperature range than that by the wild-type. Hop-21(22)ene is not produced
T41A
-
production of hop-22(29)-ene is less throughout the entire temperature range than that by the wild-type. Hop-21(22)ene is not produced
W133A
-
production of hop-22(29)-ene is less throughout the entire temperature range than that by the wild-type. Hop-21(22)ene is not produced
Y267A
-
production of hop-22(29)-ene is decreased, production of hopanol is markedly increased at lower temperatures
Y609F
site-directed mutagenesis, the mutant shows an altered product pattern compared to the wild-type enzyme, overview
Y609F
site-directed mutagenesis, the mutant shows an altered product pattern compared to the wild-type enzyme, overview. The phenotype of Y609F mutein is contrarily described in two publications
P263A
-
the mutant produces hopanol as the main product instead of hop-22(29)-ene. The mutant also produces hop-21(22)ene
P263A
-
the mutation results in a greatly enhanced production of hopanol along with the decreased formation of hopene. A high production of hopanol would be explained as follows. The point mutations could give rise to the perturbation around the front water. This disordered front water cannot correctly act as the catalytic base for the deprotonation reaction to form hopene, and in turn could be placed near to the final hopanyl cation, leading to a high production of hopanol without forming hopene
P263G
-
the mutant produces hopanol as the main product instead of hop-22(29)-ene. The mutant also produces hop-21(22)ene
P263G
-
the mutation results in a greatly enhanced production of hopanol along with the decreased formation of hopene. A high production of hopanol would be explained as follows. The point mutations could give rise to the perturbation around the front water. This disordered front water cannot correctly act as the catalytic base for the deprotonation reaction to form hopene, and in turn could be placed near to the final hopanyl cation, leading to a high production of hopanol without forming hopene
Q262G
-
the mutant produces hopanol as the main product instead of hop-22(29)-ene. The mutant also produces hop-21(22)ene
Q262G
-
the mutation results in a greatly enhanced production of hopanol along with the decreased formation of hopene. A high production of hopanol would be explained as follows. The point mutations could give rise to the perturbation around the front water. This disordered front water cannot correctly act as the catalytic base for the deprotonation reaction to form hopene, and in turn could be placed near to the final hopanyl cation, leading to a high production of hopanol without forming hopene
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
native and/or recombinant enzyme, SHC in vivo is a membrane-associated protein and can be solubilized from cell extracts by nonionic detergents, such as Triton X-100 or octylthioglucopyranoside
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene shc, DNA and amino acid sequence determination, expression in Escherichia coli
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Sato, T.; Kouda, M.; Hoshino, T.
Site-directed mutagenesis experiments on the putative deprotonation site of squalene-hopene cyclase from Alicyclobacillus acidocaldarius
Biosci. Biotechnol. Biochem.
68
728-738
2004
Alicyclobacillus acidocaldarius
Hoshino, T.; Nakano, S.; Kondo, T.; Sato, T.; Miyoshi, A.
Squalene-hopene cyclase: final deprotonation reaction, conformational analysis for the cyclization of (3R,S)-2,3-oxidosqualene and further evidence for the requirement of an isopropylidene moiety both for initiation of the polycyclization cascade and for the formation of the 5-membered E-ring
Org. Biomol. Chem.
2
1456-1470
2004
Alicyclobacillus acidocaldarius
Siedenburg, G.; Jendrossek, D.
Squalene-hopene cyclases
Appl. Environ. Microbiol.
77
3905-3915
2011
Alicyclobacillus acidocaldarius (P33247), Bradyrhizobium japonicum, Methylococcus capsulatus, no activity in Escherichia coli, Rhodopseudomonas palustris, Streptomyces peucetius, Tetrahymena thermophila, Zymomonas mobilis
EXTERNAL LINKS (specific for EC-Number 4.2.1.129)
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