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Information on EC 3.1.1.74 - cutinase and Organism(s) Colletotrichum gloeosporioides and UniProt Accession P11373

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     3 Hydrolases
         3.1 Acting on ester bonds
             3.1.1 Carboxylic-ester hydrolases
                3.1.1.74 cutinase
IUBMB Comments
Cutin, a polymeric structural component of plant cuticles, is a polymer of hydroxy fatty acids that are usually C16 or C18 and contain up to three hydroxy groups. The enzyme from several fungal sources also hydrolyses the p-nitrophenyl esters of hexadecanoic acid. It is however inactive towards several esters that are substrates for non-specific esterases.
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Colletotrichum gloeosporioides
UNIPROT: P11373
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Word Map
The taxonomic range for the selected organisms is: Colletotrichum gloeosporioides
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
cutinase, cutl1, cut190, fungal cutinase, thc_cut1, pet hydrolase, cutinase-like enzyme, lc-cutinase, cutinase 1, cdef1, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
cutin esterase
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
carboxylic ester hydrolysis
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
cutin hydrolase
Cutin, a polymeric structural component of plant cuticles, is a polymer of hydroxy fatty acids that are usually C16 or C18 and contain up to three hydroxy groups. The enzyme from several fungal sources also hydrolyses the p-nitrophenyl esters of hexadecanoic acid. It is however inactive towards several esters that are substrates for non-specific esterases.
CAS REGISTRY NUMBER
COMMENTARY hide
51377-41-4
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
4-nitrophenyl laurate + H2O
4-nitrophenol + laurate
show the reaction diagram
-
-
-
?
4-nitrophenyl myristate + H2O
4-nitrophenol + myristate
show the reaction diagram
-
-
-
?
4-nitrophenyl palmitate + H2O
4-nitrophenol + palmitate
show the reaction diagram
-
-
-
?
cutin + H2O
cutin monomers
show the reaction diagram
-
-
-
?
4-nitrophenyl acetate + H2O
4-nitrophenol + acetate
show the reaction diagram
-
low activity
-
-
?
4-nitrophenyl butyrate + H2O
4-nitrophenol + butyrate
show the reaction diagram
-
low activity
-
-
?
4-nitrophenyl caprylate + H2O
4-nitrophenol + caprylate
show the reaction diagram
-
high activity
-
-
?
4-nitrophenyl laurate + H2O
4-nitrophenol + laurate
show the reaction diagram
-
high activity
-
-
?
4-nitrophenyl myristate + H2O
4-nitrophenol + myristate
show the reaction diagram
-
high activity
-
-
?
cutin + H2O
cutin monomers
show the reaction diagram
-
-
-
?
additional information
?
-
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
cutin + H2O
cutin monomers
show the reaction diagram
-
-
-
?
cutin + H2O
cutin monomers
show the reaction diagram
-
-
-
?
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
slight activation at 1 mM
Na+
-
slight activation at 1 mM
additional information
-
the enzyme activity is poorly or not affected by 1 mM Li+, K+, Co2+, and Zn2+
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3-phenethylthio-1,1,1-trifluoropropan-2-one
-
3-phenylthio-1,1,1-trifluoropropan-2-one
-
Diethyl p-nitrophenyl phosphate
diethyl-p-nitrophenyl phosphate
-
Fe2+
-
slight inhibition at 1 mM
Mg2+
-
slight inhibition at 1 mM
Mn2+
-
slight inhibition at 1 mM
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
63
4-nitrophenyl acetate
-
pH 8.0, 25°C, recombinant enzyme
20
4-nitrophenyl butyrate
-
pH 8.0, 25°C, recombinant enzyme
7.24
4-nitrophenyl caprylate
-
pH 8.0, 25°C, recombinant enzyme
7.14
4-nitrophenyl laurate
-
pH 8.0, 25°C, recombinant enzyme
7.25
4-nitrophenyl myristate
-
pH 8.0, 25°C, recombinant enzyme
additional information
additional information
-
Michaelis-Menten kinetics
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.48
4-nitrophenyl acetate
-
pH 8.0, 25°C, recombinant enzyme
2.2
4-nitrophenyl butyrate
-
pH 8.0, 25°C, recombinant enzyme
7.7
4-nitrophenyl caprylate
-
pH 8.0, 25°C, recombinant enzyme
6.14
4-nitrophenyl laurate
-
pH 8.0, 25°C, recombinant enzyme
6.4
4-nitrophenyl myristate
-
pH 8.0, 25°C, recombinant enzyme
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
114
-
purified recombinant enzyme, pH 8.0, 25°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
recombinant wild-type enzyme
30
recombinant mutant N177D
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
evolution
cutinases are serine hydrolases that belong to the alpha/beta-hydrolase superfamily, which is divided into 2 eukaryotic and one prokaryotic subgroup, phylogenetic tree, overview. They possess a classical Ser-His-Asp catalytic triad, in which the catalytic serine is exposed to solvent. Because cutinases lack the hydrophobic lid that covers the active site serine in true lipases, the cutinase active site is large enough to accommodate the high-molecular-weight substrate cutin, and some of them can also hydrolyse high-molecular-weight synthetic polyesters
malfunction
specific inhibition of the enzyme blocks infectivity in several pathogen/host systems
physiological function
role of cutinase in the infection of plants by fungi. Fungal spores landing on the plant cuticle respond to cutin monomers by expressing cutinase
additional information
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
CUTI1_COLGL
224
0
23477
Swiss-Prot
Secretory Pathway (Reliability: 1)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
21010
21030
21170
after preincubation with 20 mM diethyl p-nitrophenyl phosphate, the peak at 21168 Da represents the covalently modified wild-type cutinase, mass spectrometry
39000
x * 39000, recombinant His-tagged enzyme, SDS-PAGE
25000
-
x * 25000, SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 39000, recombinant His-tagged enzyme, SDS-PAGE
?
-
x * 25000, SDS-PAGE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystallization and preliminary X-ray analysis of cutinase-inhibitor complexes, resolution beyond 1.6 A
in the absence and in the presence of the inhibitors diethyl p-nitrophenyl phosphate (belongs to space group P21) and 3-phenethylthio-1,1,1-trifluoropropan-2-one (belongs to space group P212121), to resolutions of 2.6 and 2.3 A, respectively. Apo-cutinase, 1.9 A resolution, belongs to space group P41212 with one subunit in the asymmetric unit with unit cell parameters a = 60, b = 60, c = 86 A, respectively. The catalytic triad (Ser136, Asp191, and His204) adopts an unusual configuration with the putative essential histidine His204 swung out of the active site into a position where it is unable to participate in catalysis, with the imidazole ring 11 A away from its expected position
PDB IDs: two inhibited structures 3DEA and 3DD5 and one uninhibited structure 3DCN
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H204N
L172K
site-directed mutagenesis, compared to the wild-type enzyme, the mutant exhibits higher enzymatic performance towards phenyl ester substrates of longer carbon chain length, yet its thermal stability is inversely affected
N177D
site-directed mutagenesis, the mutation aims to alter the surface electrostatics as well as to remove a potentially deamidation-prone asparagine residue. The mutant is more resilient to temperature increase with a 2.7fold increase in half-life at 50°C, accompanied by an increase in optimal temperature, as compared with wild-type enzyme, while the activity at 25°C is not compromised
N177D/L172K
site-directed mutagenesis, the double mutant shows enhanced activity towards phenyl ester substrates and enhanced thermal stability
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 11
-
recombinant enzyme, 25°C, stable at
730094
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50
purified wild-type enzyme and mutant L172K retain 10% activity after 2 h, mutant N177D retains 30% activity after 2 h with half-life of 30 min
50
-
recombinant enzyme, pH 8.0, 30 min, stable at
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
by ion-exchange chromatography
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain Origami B (DE3) by nickel affinity chromatography, tag cleavage by recombinant enterokinase, and anion exchange chromatography
recombinant enzyme partially 293fold from Pichia pastoris strain X-33 by ultrafiltration
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
cloned and overexpressed in Escherichia coli
expressed in Escherichia coli strain Origami B(DE3)
mutant H204N overexpressed in Escherichia coli Origami B(DE3)
sequence comparisons, functional recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain Origami B (DE3)
high-level overexpression of Glomerella cingulata cutinase, with a cutinase production of 3800 mg/l and an activity of 434 U/ml, in dense cultures of Pichia pastoris strain X-33 grown under fed-batch conditions, method evaluation, overview
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
the recombinant enzyme is induced with 0.5% v/v methanol in basal salt medium at pH 5.0 and 28°C
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Nyon, M.P.; Rice, D.W.; Berrisford, J.M.; Huang, H.; Moir, A.J.; Craven, C.J.; Nathan, S.; Mahadi, N.M.; Abu Bakar, F.D.
Crystallization and preliminary X-ray analysis of recombinant Glomerella cingulata cutinase
Acta Crystallogr. Sect. F
64
504-508
2008
Colletotrichum gloeosporioides (P11373), Colletotrichum gloeosporioides
Manually annotated by BRENDA team
Nyon, M.P.; Rice, D.W.; Berrisford, J.M.; Hounslow, A.M.; Moir, A.J.; Huang, H.; Nathan, S.; Mahadi, N.M.; Bakar, F.D.; Craven, C.J.
Catalysis by Glomerella cingulata cutinase requires conformational cycling between the active and inactive states of its catalytic triad
J. Mol. Biol.
385
226-235
2009
Fusarium solani (P00590), Fusarium solani, Colletotrichum gloeosporioides (P11373), Colletotrichum gloeosporioides
Manually annotated by BRENDA team
Chen, S.; Su, L.; Chen, J.; Wu, J.
Cutinase: characteristics, preparation, and application
Biotechnol. Adv.
31
1754-1767
2013
Alternaria brassicicola, Alternaria consortialis, Aspergillus nidulans (Q5AVY9), Aspergillus niger, Aspergillus oryzae (P52956), Bipolaris maydis, Botrytis cinerea (Q00298), Colletotrichum gloeosporioides, Colletotrichum gloeosporioides (P11373), Coprinopsis cinerea (B9U443), Cryptococcus sp. (in: Fungi) (Q874E9), Cryptococcus sp. (in: Fungi) S-2 (Q874E9), Fusarium oxysporum, Fusarium sambucinum, Fusarium solani (P00590), Helminthosporium sativum, Humicola insolens, Moesziomyces antarcticus (M9M134), Monilinia fructicola (Q2VF46), Penicillium citrinum, Penicillium sp., Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas putida, Pyrenopeziza brassicae (Q9Y7G8), Pyricularia grisea (P30272), Rhizoctonia solani, Streptomyces acidiscabies, Streptomyces badius, Streptomyces scabiei, Thermoactinomyces vulgaris, Thermobifida alba (E9LVH7), Thermobifida cellulosilytica (E9LVH8), Thermobifida cellulosilytica (E9LVH9), Thermobifida fusca, Thermobifida fusca DSM 44342, Thermothielavioides terrestris, Venturia inaequalis
Manually annotated by BRENDA team
Seman, W.M.; Bakar, S.A.; Bukhari, N.A.; Gaspar, S.M.; Othman, R.; Nathan, S.; Mahadi, N.M.; Jahim, J.; Murad, A.M.; Bakar, F.D.
High level expression of Glomerella cingulata cutinase in dense cultures of Pichia pastoris grown under fed-batch conditions
J. Biotechnol.
184
219-228
2014
Colletotrichum gloeosporioides
Manually annotated by BRENDA team
Chin, I.S.; Abdul Murad, A.M.; Mahadi, N.M.; Nathan, S.; Abu Bakar, F.D.
Thermal stability engineering of Glomerella cingulata cutinase
Protein Eng. Des. Sel.
26
369-375
2013
Colletotrichum gloeosporioides (P11373), Colletotrichum gloeosporioides
Manually annotated by BRENDA team