PETH_UNKP
ID PETH_UNKP Reviewed; 293 AA.
AC G9BY57;
DT 12-AUG-2020, integrated into UniProtKB/Swiss-Prot.
DT 22-FEB-2012, sequence version 1.
DT 03-AUG-2022, entry version 35.
DE RecName: Full=Leaf-branch compost cutinase {ECO:0000303|PubMed:24593046};
DE Short=LC-cutinase {ECO:0000303|PubMed:22194294};
DE Short=LCC {ECO:0000303|PubMed:24593046};
DE EC=3.1.1.74 {ECO:0000269|PubMed:22194294};
DE AltName: Full=PET-digesting enzyme;
DE AltName: Full=Poly(ethylene terephthalate) hydrolase {ECO:0000305|PubMed:22194294};
DE Short=PET hydrolase;
DE Short=PETase;
DE EC=3.1.1.101 {ECO:0000269|PubMed:22194294};
DE Flags: Precursor;
OS Unknown prokaryotic organism.
OC Bacteria; environmental samples.
OX NCBI_TaxID=2725;
RN [1]
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA], FUNCTION, CATALYTIC ACTIVITY, SUBSTRATE
RP SPECIFICITY, BIOPHYSICOCHEMICAL PROPERTIES, SUBCELLULAR LOCATION, ACTIVITY
RP REGULATION, AND MUTAGENESIS OF SER-165.
RX PubMed=22194294; DOI=10.1128/aem.06725-11;
RA Sulaiman S., Yamato S., Kanaya E., Kim J.J., Koga Y., Takano K., Kanaya S.;
RT "Isolation of a novel cutinase homolog with polyethylene terephthalate-
RT degrading activity from leaf-branch compost by using a metagenomic
RT approach.";
RL Appl. Environ. Microbiol. 78:1556-1562(2012).
RN [2] {ECO:0007744|PDB:4EB0}
RP X-RAY CRYSTALLOGRAPHY (1.50 ANGSTROMS) OF 36-293, FUNCTION, CATALYTIC
RP ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, DISULFIDE BOND, AND ACTIVE SITE.
RX PubMed=24593046; DOI=10.1021/bi401561p;
RA Sulaiman S., You D.J., Kanaya E., Koga Y., Kanaya S.;
RT "Crystal structure and thermodynamic and kinetic stability of metagenome-
RT derived LC-cutinase.";
RL Biochemistry 53:1858-1869(2014).
RN [3] {ECO:0007744|PDB:6THS, ECO:0007744|PDB:6THT}
RP X-RAY CRYSTALLOGRAPHY (1.10 ANGSTROMS) OF 36-293 OF MUTANTS ALA-165 AND
RP ILE-243/CYS-238/CYS-283/GLY-127, FUNCTION, CATALYTIC ACTIVITY,
RP BIOTECHNOLOGY, PROTEIN ENGINEERING, MUTAGENESIS STUDY, MUTAGENESIS OF
RP PHE-243, BIOPHYSICOCHEMICAL PROPERTIES, DISULFIDE BOND, AND ACTIVE SITE.
RX PubMed=32269349; DOI=10.1038/s41586-020-2149-4;
RA Tournier V., Topham C.M., Gilles A., David B., Folgoas C., Moya-Leclair E.,
RA Kamionka E., Desrousseaux M.L., Texier H., Gavalda S., Cot M., Guemard E.,
RA Dalibey M., Nomme J., Cioci G., Barbe S., Chateau M., Andre I.,
RA Duquesne S., Marty A.;
RT "An engineered PET depolymerase to break down and recycle plastic
RT bottles.";
RL Nature 580:216-219(2020).
CC -!- FUNCTION: Catalyzes the hydrolysis of cutin, a polyester that forms the
CC structure of plant cuticle (PubMed:22194294). Shows esterase activity
CC towards p-nitrophenol-linked aliphatic esters (pNP-aliphatic esters),
CC with a preference for short-chain substrates (C4 substrate at most)
CC (PubMed:22194294, PubMed:24593046). Cannot hydrolyze olive oil
CC (PubMed:22194294). Is also able to degrade poly(ethylene
CC terephthalate), the most abundant polyester plastic in the world
CC (PubMed:22194294, PubMed:32269349). Can also depolymerize poly(epsilon-
CC caprolactone) (PCL), a synthetic aliphatic biodegradable polyester
CC (PubMed:22194294). {ECO:0000269|PubMed:22194294,
CC ECO:0000269|PubMed:24593046, ECO:0000269|PubMed:32269349}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=cutin + H2O = cutin monomers.; EC=3.1.1.74;
CC Evidence={ECO:0000269|PubMed:22194294};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=(ethylene terephthalate)(n) + H2O = (ethylene
CC terephthalate)(n-1) + 4-[(2-hydroxyethoxy)carbonyl]benzoate + H(+);
CC Xref=Rhea:RHEA:49528, Rhea:RHEA-COMP:12420, Rhea:RHEA-COMP:12421,
CC ChEBI:CHEBI:15377, ChEBI:CHEBI:15378, ChEBI:CHEBI:131701,
CC ChEBI:CHEBI:131704; EC=3.1.1.101;
CC Evidence={ECO:0000269|PubMed:22194294, ECO:0000269|PubMed:32269349};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=4-[(2-hydroxyethoxy)carbonyl]benzoate + H2O = ethylene glycol
CC + H(+) + terephthalate; Xref=Rhea:RHEA:49532, ChEBI:CHEBI:15377,
CC ChEBI:CHEBI:15378, ChEBI:CHEBI:30043, ChEBI:CHEBI:30742,
CC ChEBI:CHEBI:131704; Evidence={ECO:0000269|PubMed:22194294,
CC ECO:0000269|PubMed:32269349};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=an acetyl ester + H2O = acetate + an aliphatic alcohol + H(+);
CC Xref=Rhea:RHEA:12957, ChEBI:CHEBI:2571, ChEBI:CHEBI:15377,
CC ChEBI:CHEBI:15378, ChEBI:CHEBI:30089, ChEBI:CHEBI:47622;
CC Evidence={ECO:0000269|PubMed:22194294};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=a butanoate ester + H2O = an aliphatic alcohol + butanoate +
CC H(+); Xref=Rhea:RHEA:47348, ChEBI:CHEBI:2571, ChEBI:CHEBI:15377,
CC ChEBI:CHEBI:15378, ChEBI:CHEBI:17968, ChEBI:CHEBI:50477;
CC Evidence={ECO:0000269|PubMed:22194294, ECO:0000269|PubMed:24593046};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=a hexanoate ester + H2O = an aliphatic alcohol + H(+) +
CC hexanoate; Xref=Rhea:RHEA:47352, ChEBI:CHEBI:2571, ChEBI:CHEBI:15377,
CC ChEBI:CHEBI:15378, ChEBI:CHEBI:17120, ChEBI:CHEBI:87656;
CC Evidence={ECO:0000269|PubMed:22194294};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=an octanoate ester + H2O = an aliphatic alcohol + H(+) +
CC octanoate; Xref=Rhea:RHEA:47356, ChEBI:CHEBI:2571, ChEBI:CHEBI:15377,
CC ChEBI:CHEBI:15378, ChEBI:CHEBI:25646, ChEBI:CHEBI:87657;
CC Evidence={ECO:0000269|PubMed:22194294};
CC -!- ACTIVITY REGULATION: Is inhibited in vitro by diethyl pNP-phosphate
CC (E600), but not by EDTA. {ECO:0000269|PubMed:22194294}.
CC -!- BIOPHYSICOCHEMICAL PROPERTIES:
CC Kinetic parameters:
CC KM=0.22 mM for pNP-butanoate (at 30 degrees Celsius and pH 8.0)
CC {ECO:0000269|PubMed:24593046};
CC KM=0.21 mM for pNP-butanoate (at 50 degrees Celsius and pH 8.0)
CC {ECO:0000269|PubMed:24593046};
CC KM=0.24 mM for pNP-butanoate (at 70 degrees Celsius and pH 8.0)
CC {ECO:0000269|PubMed:24593046};
CC Note=kcat is 232 sec(-1) with pNP-butanoate as substrate (at 30
CC degrees Celsius and pH 8.0). kcat is 343 sec(-1) with pNP-butanoate
CC as substrate (at 50 degrees Celsius and pH 8.0). kcat is 318 sec(-1)
CC with pNP-butanoate as substrate (at 70 degrees Celsius and pH 8.0).
CC {ECO:0000269|PubMed:24593046};
CC pH dependence:
CC Optimum pH is 8.5 with pNP-butyrate as substrate. Shows 70% of the
CC maximal activity at pH 7.0 and pH 9.5. {ECO:0000269|PubMed:22194294};
CC Temperature dependence:
CC Optimum temperature is 50 degrees Celsius with pNP-butanoate as
CC substrate (PubMed:22194294, PubMed:24593046). Optimum temperature
CC using PET as substrate is superior to 70 degrees Celsius
CC (PubMed:22194294). Shows 70% of the maximal activity at 30 and 70
CC degrees Celsius with pNP-butyrate as substrate. Has half-lives of 5
CC hours at 50 degrees Celsius, 80 minutes at 60 degrees Celsius, 40
CC minutes at 70 degrees Celsius and 7 minutes at 80 degrees Celsius
CC (PubMed:22194294). Is thermostable, with a determined melting
CC temperature (Tm) of 84.7 degrees Celsius (PubMed:32269349).
CC {ECO:0000269|PubMed:22194294, ECO:0000269|PubMed:24593046,
CC ECO:0000269|PubMed:32269349};
CC -!- SUBCELLULAR LOCATION: Secreted {ECO:0000305|PubMed:22194294}.
CC -!- PTM: The disulfide bond between Cys-275 and Cys-292 contributes not
CC only to the thermodynamic stability but also to the kinetic stability
CC of the enzyme. {ECO:0000269|PubMed:24593046}.
CC -!- BIOTECHNOLOGY: The engineered enzyme with mutations Ile-243/Cys-
CC 238/Cys-283/Gly-127 can hydrolyze PET in a highly efficient way,
CC ultimately achieving, in less than 10 hours, a minimum of 90 per cent
CC PET depolymerization into monomers, with a productivity of 16.7 grams
CC of terephthalate per liter per hour. It was also shown that
CC biologically recycled PET produced from enzymatically depolymerized PET
CC waste exhibits the same properties as petrochemical PET, before being
CC processed into bottles, thereby contributing towards the concept of a
CC circular PET economy. {ECO:0000269|PubMed:32269349}.
CC -!- MISCELLANEOUS: The gene encoding this enzyme comes from a leaf-branch
CC compost metagenome. The source organism remains to be identified.
CC However, it is presumably a thermophilic bacterium.
CC {ECO:0000303|PubMed:22194294}.
CC -!- SIMILARITY: Belongs to the AB hydrolase superfamily. {ECO:0000305}.
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DR EMBL; HQ704839; AEV21261.1; -; Genomic_DNA.
DR PDB; 4EB0; X-ray; 1.50 A; A=36-293.
DR PDB; 6THS; X-ray; 1.10 A; A=36-293.
DR PDB; 6THT; X-ray; 1.14 A; A=36-293.
DR PDB; 7VVC; X-ray; 1.82 A; A/B=36-293.
DR PDB; 7VVE; X-ray; 1.98 A; A/B=36-293.
DR PDB; 7W1N; X-ray; 1.88 A; A=36-293.
DR PDB; 7W44; X-ray; 1.85 A; A/B=35-293.
DR PDB; 7W45; X-ray; 1.94 A; A/B=36-293.
DR PDBsum; 4EB0; -.
DR PDBsum; 6THS; -.
DR PDBsum; 6THT; -.
DR PDBsum; 7VVC; -.
DR PDBsum; 7VVE; -.
DR PDBsum; 7W1N; -.
DR PDBsum; 7W44; -.
DR PDBsum; 7W45; -.
DR AlphaFoldDB; G9BY57; -.
DR SMR; G9BY57; -.
DR ESTHER; 9bact-g9by57; Polyesterase-lipase-cutinase.
DR BioCyc; MetaCyc:MON-21180; -.
DR SABIO-RK; G9BY57; -.
DR GO; GO:0005576; C:extracellular region; IEA:UniProtKB-SubCell.
DR GO; GO:0008126; F:acetylesterase activity; IEA:RHEA.
DR GO; GO:0050525; F:cutinase activity; IEA:UniProtKB-EC.
DR Gene3D; 3.40.50.1820; -; 1.
DR InterPro; IPR029058; AB_hydrolase.
DR InterPro; IPR029059; AB_hydrolase_5.
DR Pfam; PF12695; Abhydrolase_5; 1.
DR SUPFAM; SSF53474; SSF53474; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Disulfide bond; Hydrolase; Secreted; Serine esterase; Signal.
FT SIGNAL 1..34
FT /evidence="ECO:0000255"
FT CHAIN 35..293
FT /note="Leaf-branch compost cutinase"
FT /id="PRO_5003520086"
FT ACT_SITE 165
FT /note="Nucleophile"
FT /evidence="ECO:0000305|PubMed:24593046,
FT ECO:0000305|PubMed:32269349"
FT ACT_SITE 210
FT /note="Charge relay system"
FT /evidence="ECO:0000305|PubMed:24593046,
FT ECO:0000305|PubMed:32269349"
FT ACT_SITE 242
FT /note="Charge relay system"
FT /evidence="ECO:0000305|PubMed:24593046,
FT ECO:0000305|PubMed:32269349"
FT BINDING 95
FT /ligand="poly(ethylene terephthalate)"
FT /ligand_id="ChEBI:CHEBI:131701"
FT /evidence="ECO:0000250|UniProtKB:A0A0K8P6T7"
FT BINDING 166
FT /ligand="poly(ethylene terephthalate)"
FT /ligand_id="ChEBI:CHEBI:131701"
FT /evidence="ECO:0000250|UniProtKB:A0A0K8P6T7"
FT BINDING 190
FT /ligand="poly(ethylene terephthalate)"
FT /ligand_id="ChEBI:CHEBI:131701"
FT /evidence="ECO:0000250|UniProtKB:A0A0K8P6T7"
FT DISULFID 275..292
FT /evidence="ECO:0000269|PubMed:24593046,
FT ECO:0000269|PubMed:32269349, ECO:0007744|PDB:4EB0,
FT ECO:0007744|PDB:6THS, ECO:0007744|PDB:6THT"
FT MUTAGEN 165
FT /note="S->A: Loss of catalytic activity."
FT /evidence="ECO:0000269|PubMed:22194294"
FT MUTAGEN 243
FT /note="F->I,W: Increased PET-depolymerization activity."
FT /evidence="ECO:0000269|PubMed:32269349"
FT HELIX 48..51
FT /evidence="ECO:0007829|PDB:6THS"
FT STRAND 55..63
FT /evidence="ECO:0007829|PDB:6THS"
FT HELIX 65..67
FT /evidence="ECO:0007829|PDB:6THS"
FT STRAND 69..71
FT /evidence="ECO:0007829|PDB:6THS"
FT STRAND 74..81
FT /evidence="ECO:0007829|PDB:6THS"
FT STRAND 86..92
FT /evidence="ECO:0007829|PDB:6THS"
FT HELIX 99..102
FT /evidence="ECO:0007829|PDB:6THS"
FT HELIX 103..111
FT /evidence="ECO:0007829|PDB:6THS"
FT STRAND 115..119
FT /evidence="ECO:0007829|PDB:6THS"
FT HELIX 128..144
FT /evidence="ECO:0007829|PDB:6THS"
FT HELIX 148..151
FT /evidence="ECO:0007829|PDB:6THS"
FT STRAND 154..164
FT /evidence="ECO:0007829|PDB:6THS"
FT HELIX 166..177
FT /evidence="ECO:0007829|PDB:6THS"
FT STRAND 184..188
FT /evidence="ECO:0007829|PDB:6THS"
FT STRAND 202..207
FT /evidence="ECO:0007829|PDB:6THS"
FT STRAND 211..213
FT /evidence="ECO:0007829|PDB:6THS"
FT TURN 215..218
FT /evidence="ECO:0007829|PDB:6THS"
FT HELIX 219..225
FT /evidence="ECO:0007829|PDB:6THS"
FT STRAND 232..237
FT /evidence="ECO:0007829|PDB:6THS"
FT HELIX 244..246
FT /evidence="ECO:0007829|PDB:6THS"
FT HELIX 250..264
FT /evidence="ECO:0007829|PDB:6THS"
FT HELIX 268..273
FT /evidence="ECO:0007829|PDB:6THS"
FT STRAND 282..287
FT /evidence="ECO:0007829|PDB:6THS"
FT HELIX 290..292
FT /evidence="ECO:0007829|PDB:6THS"
SQ SEQUENCE 293 AA; 31496 MW; 202216278CDCEB8E CRC64;
MDGVLWRVRT AALMAALLAL AAWALVWASP SVEAQSNPYQ RGPNPTRSAL TADGPFSVAT
YTVSRLSVSG FGGGVIYYPT GTSLTFGGIA MSPGYTADAS SLAWLGRRLA SHGFVVLVIN
TNSRFDYPDS RASQLSAALN YLRTSSPSAV RARLDANRLA VAGHSMGGGG TLRIAEQNPS
LKAAVPLTPW HTDKTFNTSV PVLIVGAEAD TVAPVSQHAI PFYQNLPSTT PKVYVELDNA
SHFAPNSNNA AISVYTISWM KLWVDNDTRY RQFLCNVNDP ALSDFRTNNR HCQ
