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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

    Pathwayi: cocaine 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)DescriptionActionsGraphical viewLength
    Binding sitei44Substrate1
    Sitei44Probably involved in activating the substrate carbonyl and the acyl enzyme for hydrolysis1
    Active sitei117Acyl-ester intermediate1 Publication1
    Binding sitei118Substrate; via amide nitrogen1
    Active sitei259Charge relay system1 Publication1
    Active sitei287Charge relay system1 Publication1

    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)DescriptionActionsGraphical viewLength
    Mutagenesisi44Y → F: Loss of activity. Has no protective effects against cocaine-induced convulsions and lethality in rat. 2 Publications1
    Mutagenesisi55Q → A or E: Decrease in activity. 1 Publication1
    Mutagenesisi117S → A: Loss of activity. Has no protective effects against cocaine-induced convulsions and lethality in rat. 2 Publications1
    Mutagenesisi117S → C: Great decrease in activity. 2 Publications1
    Mutagenesisi151W → A: Decrease in activity. 1 Publication1
    Mutagenesisi166W → A: Decrease in activity. 1 Publication1
    Mutagenesisi169L → 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 Publication1
    Mutagenesisi172T → 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 Publication1
    Mutagenesisi173G → 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 Publication1
    Mutagenesisi259D → N: Loss of activity. 1 Publication1
    Mutagenesisi261F → A: Decrease in activity. 1 Publication1
    Mutagenesisi287H → A: Loss of activity. 1 Publication1
    Mutagenesisi407L → A: Decrease in activity. 1 Publication1
    Mutagenesisi408F → A: Decrease in activity. 1 Publication1

    Chemistry databases

    DrugBankiDB03793. Benzoic Acid.

    PTM / Processingi

    Molecule processing

    Feature keyPosition(s)DescriptionActionsGraphical viewLength
    ChainiPRO_00000900001 – 574Cocaine esteraseAdd BLAST574

    Expressioni

    Inductioni

    Positively induced by cocaine.1 Publication

    Interactioni

    Subunit structurei

    Homodimer. The protein aggregates upon heat inactivation.2 Publications

    Structurei

    Secondary structure

    1574
    Legend: HelixTurnBeta strandPDB Structure known for this area
    Show more details
    Feature keyPosition(s)DescriptionActionsGraphical viewLength
    Beta strandi6 – 15Combined sources10
    Beta strandi21 – 29Combined sources9
    Beta strandi35 – 44Combined sources10
    Helixi49 – 53Combined sources5
    Turni54 – 56Combined sources3
    Helixi60 – 64Combined sources5
    Beta strandi68 – 73Combined sources6
    Turni86 – 89Combined sources4
    Helixi90 – 103Combined sources14
    Beta strandi107 – 113Combined sources7
    Helixi118 – 127Combined sources10
    Beta strandi134 – 137Combined sources4
    Beta strandi139 – 141Combined sources3
    Helixi147 – 151Combined sources5
    Helixi160 – 177Combined sources18
    Beta strandi178 – 180Combined sources3
    Helixi185 – 196Combined sources12
    Helixi199 – 203Combined sources5
    Beta strandi205 – 207Combined sources3
    Helixi212 – 217Combined sources6
    Helixi220 – 223Combined sources4
    Turni224 – 227Combined sources4
    Helixi233 – 236Combined sources4
    Helixi241 – 244Combined sources4
    Beta strandi251 – 258Combined sources8
    Helixi262 – 272Combined sources11
    Turni273 – 275Combined sources3
    Beta strandi278 – 286Combined sources9
    Beta strandi291 – 294Combined sources4
    Helixi301 – 303Combined sources3
    Helixi307 – 322Combined sources16
    Turni326 – 331Combined sources6
    Beta strandi334 – 340Combined sources7
    Turni341 – 343Combined sources3
    Beta strandi344 – 352Combined sources9
    Beta strandi357 – 364Combined sources8
    Beta strandi377 – 381Combined sources5
    Beta strandi387 – 393Combined sources7
    Beta strandi407 – 410Combined sources4
    Helixi419 – 421Combined sources3
    Beta strandi428 – 431Combined sources4
    Beta strandi439 – 457Combined sources19
    Beta strandi459 – 467Combined sources9
    Beta strandi473 – 482Combined sources10
    Helixi483 – 485Combined sources3
    Beta strandi489 – 491Combined sources3
    Beta strandi501 – 514Combined sources14
    Beta strandi519 – 526Combined sources8
    Beta strandi537 – 540Combined sources4
    Helixi542 – 544Combined sources3
    Helixi547 – 549Combined sources3
    Beta strandi553 – 563Combined sources11
    Beta strandi566 – 572Combined sources7

    3D structure databases

    Select the link destinations:
    PDBei
    RCSB PDBi
    PDBji
    Links Updated
    PDB 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.
    ModBaseiSearch...
    MobiDBiSearch...

    Miscellaneous databases

    EvolutionaryTraceiQ9L9D7.

    Family & Domainsi

    Region

    Feature keyPosition(s)DescriptionActionsGraphical viewLength
    Regioni1 – 1441AAdd BLAST144
    Regioni145 – 2402Add BLAST96
    Regioni241 – 3541BAdd BLAST114
    Regioni355 – 5743Add BLAST220

    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.

    Genome annotation databases

    KEGGiag:AAF42807.

    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
    PDB 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.
    ModBaseiSearch...
    MobiDBiSearch...

    Chemistry databases

    DrugBankiDB03793. Benzoic Acid.

    Protein family/group databases

    ESTHERirhosm-cocE. Cocaine_esterase.

    Protocols and materials databases

    Structural Biology KnowledgebaseSearch...

    Genome annotation databases

    KEGGiag:AAF42807.

    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...

    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: November 2, 2016
    This is version 98 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

    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 one 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.