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Protein

Cocaine esterase

Gene

cocE

Organism
Rhodococcus sp. (strain MB1 Bresler)
Status
Reviewed-Annotation score: Annotation score: 5 out of 5-Experimental evidence at protein leveli

Functioni

Hydrolyzes cocaine to benzoate and ecgonine methyl ester, endowing the bacteria with the ability to utilize cocaine as a sole source of carbon and energy for growth, as this bacterium lives in the rhizosphere of coca plants. Also efficiently hydrolyzes cocaethylene, a more potent cocaine metabolite that has been observed in patients who concurrently abuse cocaine and alcohol. Is able to prevent cocaine-induced convulsions and lethality in rat.3 Publications

Catalytic activityi

Cocaine + H2O = ecgonine methyl ester + benzoate.3 Publications

Kineticsi

kcat is 7.8 sec(-1) with cocaine as substrate, and 9.4 sec(-1) with cocaethylene.

  1. KM=0.64 µM for cocaine1 Publication
  2. KM=1.6 µM for cocaethylene1 Publication

    pH dependencei

    Optimum pH is 9.0.1 Publication

    Temperature dependencei

    Is relatively unstable at physiological temperatures since it displays a half-life of 13 minutes in rat plasma at 37 degrees Celsius.2 Publications

    Pathway:icocaine degradation

    This protein is involved in the pathway cocaine degradation, which is part of Alkaloid degradation.
    View all proteins of this organism that are known to be involved in the pathway cocaine degradation and in Alkaloid degradation.

    Sites

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Binding sitei44 – 441Substrate
    Sitei44 – 441Probably involved in activating the substrate carbonyl and the acyl enzyme for hydrolysis
    Active sitei117 – 1171Acyl-ester intermediate1 Publication
    Binding sitei118 – 1181Substrate; via amide nitrogen
    Active sitei259 – 2591Charge relay system1 Publication
    Active sitei287 – 2871Charge relay system1 Publication

    GO - Molecular functioni

    GO - Biological processi

    Complete GO annotation...

    Keywords - Molecular functioni

    Hydrolase, Serine esterase

    Enzyme and pathway databases

    BioCyciMetaCyc:MONOMER-15371.
    BRENDAi3.1.1.84. 5397.
    UniPathwayiUPA00110.

    Protein family/group databases

    ESTHERirhosm-cocE. Cocaine_esterase.

    Names & Taxonomyi

    Protein namesi
    Recommended name:
    Cocaine esterase (EC:3.1.1.84)
    Gene namesi
    Name:cocE
    OrganismiRhodococcus sp. (strain MB1 Bresler)
    Taxonomic identifieri104109 [NCBI]
    Taxonomic lineageiBacteriaActinobacteriaCorynebacterialesNocardiaceaeRhodococcus

    Subcellular locationi

    GO - Cellular componenti

    Complete GO annotation...

    Keywords - Cellular componenti

    Cytoplasm

    Pathology & Biotechi

    Biotechnological usei

    Because of the high catalytic proficiency of CocE, it is an attractive candidate for novel protein-based therapies for cocaine overdose, as this cocaine-degrading enzyme could be used for rapid cocaine detoxification in an emergency setting. However, wild-type CocE is relatively unstable, but this can be improved by specific mutations. Thus, improved stability of engineered CocE enzymes will have a profound influence on the use of this protein to combat cocaine-induced toxicity and addiction in humans. Has also a potential as a highly-sensitive drug detector.2 Publications

    Mutagenesis

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Mutagenesisi44 – 441Y → F: Loss of activity. Has no protective effects against cocaine-induced convulsions and lethality in rat. 2 Publications
    Mutagenesisi55 – 551Q → A or E: Decrease in activity. 1 Publication
    Mutagenesisi117 – 1171S → A: Loss of activity. Has no protective effects against cocaine-induced convulsions and lethality in rat. 2 Publications
    Mutagenesisi117 – 1171S → C: Great decrease in activity. 2 Publications
    Mutagenesisi151 – 1511W → A: Decrease in activity. 1 Publication
    Mutagenesisi166 – 1661W → A: Decrease in activity. 1 Publication
    Mutagenesisi169 – 1691L → K: Displays greatly enhanced stability, with a half-life of 570 minutes at 37 degrees Celsius. Exhibits 4.5-fold reduction in catalytic efficiency. 1 Publication
    Mutagenesisi172 – 1721T → R: Displays enhanced stability, with a half-life of 78 minutes at 37 degrees Celsius, and exhibits 3-fold reduction in catalytic efficiency. Displays enhanced stability, with a half-life of 370 minutes at 37 degrees Celsius, and exhibits 3-fold reduction in catalytic efficiency; when associated with Q-173. 1 Publication
    Mutagenesisi173 – 1731G → Q: Displays enhanced stability, with a half-life of 75 minutes at 37 degrees Celsius, and has no deleterious effect on catalytic efficiency. Displays enhanced stability, with a half-life of 370 minutes at 37 degrees Celsius, and exhibits 3-fold reduction in catalytic efficiency; when associated with R-172. 1 Publication
    Mutagenesisi259 – 2591D → N: Loss of activity. 1 Publication
    Mutagenesisi261 – 2611F → A: Decrease in activity. 1 Publication
    Mutagenesisi287 – 2871H → A: Loss of activity. 1 Publication
    Mutagenesisi407 – 4071L → A: Decrease in activity. 1 Publication
    Mutagenesisi408 – 4081F → A: Decrease in activity. 1 Publication

    PTM / Processingi

    Molecule processing

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Chaini1 – 574574Cocaine esterasePRO_0000090000Add
    BLAST

    Expressioni

    Inductioni

    Positively induced by cocaine.1 Publication

    Interactioni

    Subunit structurei

    Homodimer. The protein aggregates upon heat inactivation.2 Publications

    Structurei

    Secondary structure

    1
    574
    Legend: HelixTurnBeta strand
    Show more details
    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Beta strandi6 – 1510Combined sources
    Beta strandi21 – 299Combined sources
    Beta strandi35 – 4410Combined sources
    Helixi49 – 535Combined sources
    Turni54 – 563Combined sources
    Helixi60 – 645Combined sources
    Beta strandi68 – 736Combined sources
    Turni86 – 894Combined sources
    Helixi90 – 10314Combined sources
    Beta strandi107 – 1137Combined sources
    Helixi118 – 12710Combined sources
    Beta strandi134 – 1374Combined sources
    Beta strandi139 – 1413Combined sources
    Helixi147 – 1515Combined sources
    Helixi160 – 17718Combined sources
    Beta strandi178 – 1803Combined sources
    Helixi185 – 19612Combined sources
    Helixi199 – 2035Combined sources
    Beta strandi205 – 2073Combined sources
    Helixi212 – 2176Combined sources
    Helixi220 – 2234Combined sources
    Turni224 – 2274Combined sources
    Helixi233 – 2364Combined sources
    Helixi241 – 2444Combined sources
    Beta strandi251 – 2588Combined sources
    Helixi262 – 27211Combined sources
    Turni273 – 2753Combined sources
    Beta strandi278 – 2869Combined sources
    Beta strandi291 – 2944Combined sources
    Helixi301 – 3033Combined sources
    Helixi307 – 32216Combined sources
    Turni326 – 3316Combined sources
    Beta strandi334 – 3407Combined sources
    Turni341 – 3433Combined sources
    Beta strandi344 – 3529Combined sources
    Beta strandi357 – 3648Combined sources
    Beta strandi377 – 3815Combined sources
    Beta strandi387 – 3937Combined sources
    Beta strandi407 – 4104Combined sources
    Helixi419 – 4213Combined sources
    Beta strandi428 – 4314Combined sources
    Beta strandi439 – 45719Combined sources
    Beta strandi459 – 4679Combined sources
    Beta strandi473 – 48210Combined sources
    Helixi483 – 4853Combined sources
    Beta strandi489 – 4913Combined sources
    Beta strandi501 – 51414Combined sources
    Beta strandi519 – 5268Combined sources
    Beta strandi537 – 5404Combined sources
    Helixi542 – 5443Combined sources
    Helixi547 – 5493Combined sources
    Beta strandi553 – 56311Combined sources
    Beta strandi566 – 5727Combined sources

    3D structure databases

    Select the link destinations:
    PDBei
    RCSB PDBi
    PDBji
    Links Updated
    EntryMethodResolution (Å)ChainPositionsPDBsum
    1JU3X-ray1.58A1-574[»]
    1JU4X-ray1.63A1-574[»]
    1L7QX-ray1.76A1-574[»]
    1L7RX-ray1.64A1-574[»]
    3I2FX-ray2.50A1-574[»]
    3I2GX-ray2.50A1-574[»]
    3I2HX-ray1.65A1-574[»]
    3I2IX-ray2.14A1-574[»]
    3I2JX-ray2.01A1-574[»]
    3I2KX-ray1.51A1-574[»]
    3IDAX-ray1.60A1-574[»]
    3PUHX-ray2.30A/B1-574[»]
    3PUIX-ray1.53A1-574[»]
    4P08X-ray2.34A4-574[»]
    ProteinModelPortaliQ9L9D7.
    SMRiQ9L9D7. Positions 5-574.
    ModBaseiSearch...
    MobiDBiSearch...

    Miscellaneous databases

    EvolutionaryTraceiQ9L9D7.

    Family & Domainsi

    Region

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Regioni1 – 1441441AAdd
    BLAST
    Regioni145 – 240962Add
    BLAST
    Regioni241 – 3541141BAdd
    BLAST
    Regioni355 – 5742203Add
    BLAST

    Domaini

    It consists of three domains: domain 1 contains the active site; domains 2 and 3 are involved in substrate recognition. Domain 1 contains the GxSxxG motif found in most members of the alpha/beta hydrolase superfamily.1 Publication

    Sequence similaritiesi

    Belongs to the CocE/NonD hydrolase family.Curated

    Family and domain databases

    Gene3Di2.60.120.260. 1 hit.
    3.40.50.1820. 2 hits.
    InterProiIPR029058. AB_hydrolase.
    IPR005674. CocE/Ser_esterase.
    IPR008979. Galactose-bd-like.
    IPR000383. Xaa-Pro-like_dom.
    IPR013736. Xaa-Pro_dipept_C.
    [Graphical view]
    PfamiPF02129. Peptidase_S15. 1 hit.
    PF08530. PepX_C. 1 hit.
    [Graphical view]
    SMARTiSM00939. PepX_C. 1 hit.
    [Graphical view]
    SUPFAMiSSF49785. SSF49785. 1 hit.
    SSF53474. SSF53474. 1 hit.
    TIGRFAMsiTIGR00976. /NonD. 1 hit.

    Sequencei

    Sequence statusi: Complete.

    Q9L9D7-1 [UniParc]FASTAAdd to basket

    « Hide

            10         20         30         40         50
    MVDGNYSVAS NVMVPMRDGV RLAVDLYRPD ADGPVPVLLV RNPYDKFDVF
    60 70 80 90 100
    AWSTQSTNWL EFVRDGYAVV IQDTRGLFAS EGEFVPHVDD EADAEDTLSW
    110 120 130 140 150
    ILEQAWCDGN VGMFGVSYLG VTQWQAAVSG VGGLKAIAPS MASADLYRAP
    160 170 180 190 200
    WYGPGGALSV EALLGWSALI GTGLITSRSD ARPEDAADFV QLAAILNDVA
    210 220 230 240 250
    GAASVTPLAE QPLLGRLIPW VIDQVVDHPD NDESWQSISL FERLGGLATP
    260 270 280 290 300
    ALITAGWYDG FVGESLRTFV AVKDNADARL VVGPWSHSNL TGRNADRKFG
    310 320 330 340 350
    IAATYPIQEA TTMHKAFFDR HLRGETDALA GVPKVRLFVM GIDEWRDETD
    360 370 380 390 400
    WPLPDTAYTP FYLGGSGAAN TSTGGGTLST SISGTESADT YLYDPADPVP
    410 420 430 440 450
    SLGGTLLFHN GDNGPADQRP IHDRDDVLCY STEVLTDPVE VTGTVSARLF
    460 470 480 490 500
    VSSSAVDTDF TAKLVDVFPD GRAIALCDGI VRMRYRETLV NPTLIEAGEI
    510 520 530 540 550
    YEVAIDMLAT SNVFLPGHRI MVQVSSSNFP KYDRNSNTGG VIAREQLEEM
    560 570
    CTAVNRIHRG PEHPSHIVLP IIKR
    Length:574
    Mass (Da):62,132
    Last modified:October 1, 2000 - v1
    Checksum:i9E35724F586089B7
    GO

    Sequence databases

    Select the link destinations:
    EMBLi
    GenBanki
    DDBJi
    Links Updated
    AF173165 Genomic DNA. Translation: AAF42807.1.

    Cross-referencesi

    Web resourcesi

    Protein Spotlight

    Rhodococcus: Nature's junkie - Issue 27 of October 2002

    Sequence databases

    Select the link destinations:
    EMBLi
    GenBanki
    DDBJi
    Links Updated
    AF173165 Genomic DNA. Translation: AAF42807.1.

    3D structure databases

    Select the link destinations:
    PDBei
    RCSB PDBi
    PDBji
    Links Updated
    EntryMethodResolution (Å)ChainPositionsPDBsum
    1JU3X-ray1.58A1-574[»]
    1JU4X-ray1.63A1-574[»]
    1L7QX-ray1.76A1-574[»]
    1L7RX-ray1.64A1-574[»]
    3I2FX-ray2.50A1-574[»]
    3I2GX-ray2.50A1-574[»]
    3I2HX-ray1.65A1-574[»]
    3I2IX-ray2.14A1-574[»]
    3I2JX-ray2.01A1-574[»]
    3I2KX-ray1.51A1-574[»]
    3IDAX-ray1.60A1-574[»]
    3PUHX-ray2.30A/B1-574[»]
    3PUIX-ray1.53A1-574[»]
    4P08X-ray2.34A4-574[»]
    ProteinModelPortaliQ9L9D7.
    SMRiQ9L9D7. Positions 5-574.
    ModBaseiSearch...
    MobiDBiSearch...

    Protein family/group databases

    ESTHERirhosm-cocE. Cocaine_esterase.

    Protocols and materials databases

    Structural Biology KnowledgebaseSearch...

    Enzyme and pathway databases

    UniPathwayiUPA00110.
    BioCyciMetaCyc:MONOMER-15371.
    BRENDAi3.1.1.84. 5397.

    Miscellaneous databases

    EvolutionaryTraceiQ9L9D7.

    Family and domain databases

    Gene3Di2.60.120.260. 1 hit.
    3.40.50.1820. 2 hits.
    InterProiIPR029058. AB_hydrolase.
    IPR005674. CocE/Ser_esterase.
    IPR008979. Galactose-bd-like.
    IPR000383. Xaa-Pro-like_dom.
    IPR013736. Xaa-Pro_dipept_C.
    [Graphical view]
    PfamiPF02129. Peptidase_S15. 1 hit.
    PF08530. PepX_C. 1 hit.
    [Graphical view]
    SMARTiSM00939. PepX_C. 1 hit.
    [Graphical view]
    SUPFAMiSSF49785. SSF49785. 1 hit.
    SSF53474. SSF53474. 1 hit.
    TIGRFAMsiTIGR00976. /NonD. 1 hit.
    ProtoNetiSearch...

    Publicationsi

    1. "Gene cloning and nucleotide sequencing and properties of a cocaine esterase from Rhodococcus sp. strain MB1."
      Bresler M.M., Rosser S.J., Basran A., Bruce N.C.
      Appl. Environ. Microbiol. 66:904-908(2000) [PubMed] [Europe PMC] [Abstract]
      Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA], PROTEIN SEQUENCE OF 1-11, FUNCTION, CATALYTIC ACTIVITY, BIOTECHNOLOGY, INDUCTION.
      Strain: MB1.
    2. "Rapid and robust protection against cocaine-induced lethality in rats by the bacterial cocaine esterase."
      Cooper Z.D., Narasimhan D., Sunahara R.K., Mierzejewski P., Jutkiewicz E.M., Larsen N.A., Wilson I.A., Landry D.W., Woods J.H.
      Mol. Pharmacol. 70:1885-1891(2006) [PubMed] [Europe PMC] [Abstract]
      Cited for: FUNCTION, TEMPERATURE DEPENDENCE, MUTAGENESIS OF TYR-44 AND SER-117.
      Strain: MB1.
    3. Cited for: X-RAY CRYSTALLOGRAPHY (1.58 ANGSTROMS) IN COMPLEXES WITH BENZOATE AND TRANSITION STATE ANALOG, ACTIVE SITE, REACTION MECHANISM, DOMAIN.
      Strain: MB1.
    4. "Biochemical characterization and structural analysis of a highly proficient cocaine esterase."
      Turner J.M., Larsen N.A., Basran A., Barbas C.F. III, Bruce N.C., Wilson I.A., Lerner R.A.
      Biochemistry 41:12297-12307(2002) [PubMed] [Europe PMC] [Abstract]
      Cited for: X-RAY CRYSTALLOGRAPHY (1.6 ANGSTROMS) OF MUTANTS PHE-44 AND ALA-117 IN COMPLEX WITH BENZOATE, FUNCTION, CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, BIOTECHNOLOGY, MUTAGENESIS OF TYR-44; GLN-55; SER-117; TRP-151; TRP-166; ASP-259; PHE-261; HIS-287; LEU-407 AND PHE-408.
      Strain: MB1.
    5. Cited for: X-RAY CRYSTALLOGRAPHY (1.51 ANGSTROMS) OF WILD-TYPE AND MUTANTS LYS-169; ARG-172; GLN-173 AND ARG-172/GLN-173, MUTAGENESIS OF LEU-169; THR-172 AND GLY-173, CATALYTIC ACTIVITY, SUBUNIT.
      Strain: MB1.
    6. "Subunit stabilization and polyethylene glycolation of cocaine esterase improves in vivo residence time."
      Narasimhan D., Collins G.T., Nance M.R., Nichols J., Edwald E., Chan J., Ko M.C., Woods J.H., Tesmer J.J., Sunahara R.K.
      Mol. Pharmacol. 80:1056-1065(2011) [PubMed] [Europe PMC] [Abstract]
      Cited for: X-RAY CRYSTALLOGRAPHY (1.53 ANGSTROMS) OF WILD-TYPE AND A DISULFIDE-STABILIZED DIMERIC MUTANT.
      Strain: MB1.

    Entry informationi

    Entry nameiCOCE_RHOSM
    AccessioniPrimary (citable) accession number: Q9L9D7
    Entry historyi
    Integrated into UniProtKB/Swiss-Prot: January 31, 2002
    Last sequence update: October 1, 2000
    Last modified: June 24, 2015
    This is version 92 of the entry and version 1 of the sequence. [Complete history]
    Entry statusiReviewed (UniProtKB/Swiss-Prot)
    Annotation programProkaryotic Protein Annotation Program

    Miscellaneousi

    Miscellaneous

    This enzyme hydrolyzes cocaine faster than any other known cocaine esterase.
    Incorporating disulfide bonds between cysteine residues substituted at Gly-4 and Ser-10 conveys significant improvements to the thermostability and the half-life at 37 degrees Celsius. Moreover, in combination with T172R/G173Q mutations, the disulfide-stabilized dimer (CCRQ-CocE) remains more than 90% active for longer than 40 days at 37 degrees Celsius, representing a >4700-fold improvement over wt-CocE. The enhanced stability serves as a better substrate for modification, with polyethylene glycol (PEG) moieties providing the therapeutic with stealth properties. PEGylated CCRQ-CocE retains full in vitro enzymatic activity, protects rodents up to 72 hours in a cocaine overdose model, diminishes self-administration for 72 hours in rats, reduces cocaine-induced cardiovascular effects and locomotor functions in monkeys for up to 48 hours, and displays reduced immunogenicity in mice.

    Keywords - Technical termi

    3D-structure, Direct protein sequencing

    Documents

    1. PATHWAY comments
      Index of metabolic and biosynthesis pathways
    2. PDB cross-references
      Index of Protein Data Bank (PDB) cross-references
    3. Protein Spotlight
      Protein Spotlight articles and cited UniProtKB/Swiss-Prot entries
    4. SIMILARITY comments
      Index of protein domains and families

    External Data

    Dasty 3

    Similar proteinsi

    Links to similar proteins from the UniProt Reference Clusters (UniRef) at 100%, 90% and 50% sequence identity:
    100%UniRef100 combines identical sequences and sub-fragments with 11 or more residues from any organism into Uniref entry.
    90%UniRef90 is built by clustering UniRef100 sequences that have at least 90% sequence identity to, and 80% overlap with, the longest sequence (a.k.a seed sequence).
    50%UniRef50 is built by clustering UniRef90 seed sequences that have at least 50% sequence identity to, and 80% overlap with, the longest sequence in the cluster.