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Information on EC 3.1.1.74 - cutinase and Organism(s) Aspergillus oryzae and UniProt Accession P52956

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EC Tree
     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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Aspergillus oryzae
UNIPROT: P52956
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The taxonomic range for the selected organisms is: Aspergillus oryzae
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
-
-
-
-
cutin hydrolase
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
carboxylic ester hydrolysis
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-
-
-
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 acetate + H2O
4-nitrophenol + acetate
show the reaction diagram
-
-
-
?
4-nitrophenyl butyrate + H2O
4-nitrophenol + butyrate
show the reaction diagram
4-nitrophenyl hexanoate + H2O
4-nitrophenol + hexanoate
show the reaction diagram
-
-
-
?
4-nitrophenyl valerate + H2O
4-nitrophenol + valerate
show the reaction diagram
-
-
-
?
cutin + H2O
cutin monomers
show the reaction diagram
poly(epsilon-caprolactone) + H2O
?
show the reaction diagram
-
-
-
?
4-nitrophenyl acetate + H2O
4-nitrophenol + acetate
show the reaction diagram
-
-
-
-
?
4-nitrophenyl butyrate + H2O
4-nitrophenol + butyrate
show the reaction diagram
-
-
-
-
?
4-nitrophenyl hexanoate
4-nitrophenol + hexanoate
show the reaction diagram
-
-
-
-
?
4-nitrophenyl valerate
4-nitrophenol + pentanoate
show the reaction diagram
-
-
-
-
?
polybutylene succinate co-adipate + H2O
?
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
cutinases perform their catalysis in two discrete steps, with a covalent intermediate that links the catalytic serine to the carbonyl group of the ester being hydrolyzed
-
-
?
additional information
?
-
cutinases are capable of catalyzing esterification and transesterification
-
-
?
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
59.2
4-nitrophenyl butyrate
pH and temperature not specified in the publication
55.3
4-nitrophenyl valerate
pH and temperature not specified in the publication
0.00496
4-nitrophenyl acetate
-
in 14.5 mM Tris-HCl buffer, pH 7.5, 0.75% glycerol
0.00021 - 0.029
4-nitrophenyl butyrate
0.00029
4-nitrophenyl hexanoate
-
in 14.5 mM Tris-HCl buffer, pH 7.5, 0.75% glycerol
0.00004
4-nitrophenyl valerate
-
in 14.5 mM Tris-HCl buffer, pH 7.5, 0.75% glycerol
additional information
additional information
-
comparison of kinetics of cutinases from different organisms, overview
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0163
4-nitrophenyl butyrate
-
pH 7.5, 25°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00000167
4-nitrophenyl acetate
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in 14.5 mM Tris-HCl buffer, pH 7.5, 0.75% glycerol
0.0000582 - 0.0567
4-nitrophenyl butyrate
0.000023
4-nitrophenyl hexanoate
-
in 14.5 mM Tris-HCl buffer, pH 7.5, 0.75% glycerol
0.000053
4-nitrophenyl valerate
-
in 14.5 mM Tris-HCl buffer, pH 7.5, 0.75% glycerol
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5 - 8
-
assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 9
-
has activity superior to lipases
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
assay at
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
the mutant enzyme Q110W/K114W is retained in the endoplasmic reticulum whereas wild-type enzyme is secreted
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
physiological function
additional information
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
-
crystallography
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
PDB IDs 3GBS, 3PQD, X-ray diffraction structure determination at 1.75 A resolution
to 1.75 A resolution. Alpha/beta fold hallmarked by a central beta-sheet of 5 parallel strands surrounded by 10 alpha-helices. An additional disulfide bond and a topologically favored catalytic triad (Ser126, Asp181, and His194), with a continuous and deep groove
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A102D/Q105R/G106E
pH-optima for activity and stability are identical to wild-type enzym. Improvement in Tm-value of 3.4°C. Increased half-life at 6°C relative to the wild-type enzyme of approximately 3fold
A102D/Q105R/G106E/N133A/S140P/E161T/A166P
large improvement of stability at 60°C
A102D/Q105R/G106E/N133A/S140P/E161T/A166P/K137E
large improvement of stability at 60°C
A102D/Q105R/G106E/Q98N/A99D/E109Q
thermodynamically most stable variant, improving on wild-type enzyme by 6.7 kJ/mol
A178P/V179P
loss of stability and activity
K174R/Y176F/A178E/D200R/G202E/D203E/D206R
mutant enzyme shows an increased kinetic stability
L26D/G28E/D30R/K67R
improvement in Tm-value of 0.7°C
N133A/S140P/E161T/A166P
proline mutations contribute to themostabilization by decreasing the entropy lost upon folding. Improvement in Tm of 1.7°C. Increased half-life at 6°C relative to the wild-type enzyme of approximately 2fold
Q110W/K114W
the mutant enzyme is retained in the endoplasmic reticulum whereas wild-type enzyme is secreted
R46P
the Tm-value is 3°C below that of wild-type enzyme
T84R/D86L/A99E/A100S
decrease in thermostability relative to the wild-type enzyme. Large losses in 4-nitrophenyl butyrate (about 70%) and poly(epsilon-caprolactone) (about 90%) activities
V150I/I136V
mutation do not provide any improvement in stability
D30S
mutation increases the KD value for interaction with hydrophobin RolA
D30S/E31S/D142S/D171S
mutation D30S increases the KD value for interaction with hydrophobin RolA in comparison with mutant E31S/D142S/D171S
additional information
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
the enzyme is unstable and functions poorly at high temperatures as well as at acidic pH conditions, biophysical parameters of cutinase as a function of pH, overview
713927
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
t1/2-value: 4.5 h (wild-type enzyme), 40 h (mutant enzymes A102D/Q105R/G106E/N133A/S140P/E161T/A166P and 102D/Q105R/G106E/N133A/S140P/E161T/A166P/K137E), 11.2 h (mutant enzyme A102D/Q105R/G106E/Q98N/A99D/E109Q)
65
t1/2-value: 10 min (wild-type enzyme), 4 h (mutant enzyme A102D/Q105R/G106E/N133A/S140P/E161T/A166P), 5 h (mutant enzyme A102D/Q105R/G106E/N133A/S140P/E161T/A166P/K137E)
59
-
melting temperature. Maintains a high level of activity at 40 °C
additional information
-
the enzyme is unstable and functions poorly at high temperatures as well as at acidic pH conditions, differential scanning calorimetry thermograms of cutinase, overview
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Saccharomyces cerevisiae
heterologously expressed in Pichia pastoris using the strong methanol-induced AOX I promoter. The protein is expressed with the Saccharomyces cerevisiae alpha factor tag on the N-terminal for extracellular secretion and the six histidine tag on the C-terminus for ease of purification using affinity chromatography
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
expression of enzyme in Saccharomyces cerevisiae. Approximately 28% of the cutinase localizes to the cell walls and/or between cell wall and cell membrane. Protoplasts entrapped in a membrane capsule with a low-viscous liquid-core of 1.92 % w/v calcium-alginate in a static culture secrete measurable amounts of cutinase into the broth. The entrapped protoplasts are cultivated in a shake flask at low osmotic pressure without disruption. During 60 h of cultivation, the extracellular cutinase activity of the free protoplasts at 29.3 atm and protoplasts entrapped in the capsule at 17.2 atm are 0.13 and 0.39 U/mL, respectively
biotechnology
-
engineering new cutinase-inspired biocatalysts with tailor-made properties
degradation
-
biotechnological applications of cutinases for synthetic polyester degradation
industry
-
useful as biocatalysts in systems involving hydrolysis, esterification, and transesterification reactions. Displays a stability profile that is well-fitted to the industrial process
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Pio, T.; Macedo, G.
Cutinases: Properties and industrial applications
Adv. Appl. Microbiol.
66
77-95
2009
Aspergillus oryzae, Fusarium oxysporum, Fusarium solani, Humicola insolens
Manually annotated by BRENDA team
Liu, Z.; Gosser, Y.; Baker, P.J.; Ravee, Y.; Lu, Z.; Alemu, G.; Li, H.; Butterfoss, G.L.; Kong, X.P.; Gross, R.; Montclare, J.K.
Structural and functional studies of Aspergillus oryzae cutinase: enhanced thermostability and hydrolytic activity of synthetic ester and polyester degradation
J. Am. Chem. Soc.
131
15711-15716
2009
Aspergillus oryzae, Fusarium solani
Manually annotated by BRENDA team
Baker, P.; Poultney, C.; Liu, Z.; Gross, R.; Montclare, J.
Identification and comparison of cutinases for synthetic polyester degradation
Appl. Microbiol. Biotechnol.
93
229-240
2012
Aspergillus fumigatus, Aspergillus oryzae, Fusarium solani, Humicola insolens, Alternaria brassicicola
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
Terauchi, Y.; Kim, Y.K.; Tanaka, T.; Nanatani, K.; Takahashi, T.; Abe, K.
Asp30 of Aspergillus oryzae cutinase CutL1 is involved in the ionic interaction with fungal hydrophobin RolA
Biosci. Biotechnol. Biochem.
81
1363-1368
2017
Aspergillus oryzae (I7GSC4), Aspergillus oryzae
Manually annotated by BRENDA team
Takahashi, T.; Tanaka, T.; Tsushima, Y.; Muragaki, K.; Uehara, K.; Takeuchi, S.; Maeda, H.; Yamagata, Y.; Nakayama, M.; Yoshimi, A.; Abe, K.
Ionic interaction of positive amino acid residues of fungal hydrophobin RolA with acidic amino acid residues of cutinase CutL1
Mol. Microbiol.
96
14-27
2015
Aspergillus oryzae (I7GSC4), Aspergillus oryzae
Manually annotated by BRENDA team
Shirke, A.N.; Basore, D.; Butterfoss, G.L.; Bonneau, R.; Bystroff, C.; Gross, R.A.
Toward rational thermostabilization of Aspergillus oryzae cutinase Insights into catalytic and structural stability
Proteins
84
60-72
2016
Aspergillus oryzae (P52956), Aspergillus oryzae, Aspergillus oryzae ATCC 42149 (P52956)
Manually annotated by BRENDA team
Aoyagi, H.; Katakura, Y.; Iwasaki, A.
Production of secretory cutinase by recombinant Saccharomyces cerevisiae protoplasts
SpringerPlus
5
1-6
2016
Aspergillus oryzae (P52956), Aspergillus oryzae ATCC 42149 (P52956)
Manually annotated by BRENDA team