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   27-MAR-2024, entry version 39.
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   PDB; 8JMO; X-ray; 1.95 A; A/B=36-293.
DR   PDB; 8JMP; X-ray; 1.90 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   PDBsum; 8JMO; -.
DR   PDBsum; 8JMP; -.
DR   AlphaFoldDB; G9BY57; -.
DR   SMR; G9BY57; -.
DR   ESTHER; 9bact-g9by57; Polyesterase-lipase-cutinase.
DR   BioCyc; MetaCyc:MONOMER-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; alpha/beta hydrolase; 1.
DR   InterPro; IPR029058; AB_hydrolase.
DR   InterPro; IPR029059; AB_hydrolase_5.
DR   PANTHER; PTHR22946:SF9; POLYKETIDE TRANSFERASE AF380; 1.
DR   PANTHER; PTHR22946; UNCHARACTERIZED; 1.
DR   Pfam; PF12695; Abhydrolase_5; 1.
DR   SUPFAM; SSF53474; alpha/beta-Hydrolases; 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
//