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Q9L9D7

- COCE_RHOSM

UniProt

Q9L9D7 - COCE_RHOSM

Protein

Cocaine esterase

Gene

cocE

Organism
Rhodococcus sp. (strain MB1 Bresler)
Status
Reviewed - Annotation score: 5 out of 5- Experimental evidence at protein leveli
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    • History
      Entry version 86 (01 Oct 2014)
      Sequence version 1 (01 Oct 2000)
      Previous versions | rss
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    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

    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

    1. dipeptidyl-peptidase activity Source: InterPro

    GO - Biological processi

    1. cocaine catabolic process Source: UniProtKB-UniPathway

    Keywords - Molecular functioni

    Hydrolase, Serine esterase

    Enzyme and pathway databases

    BioCyciMetaCyc:MONOMER-15371.
    BRENDAi3.1.1.1. 5397.
    UniPathwayiUPA00110.

    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 lineageiBacteriaActinobacteriaActinobacteridaeActinomycetalesCorynebacterineaeNocardiaceaeRhodococcus

    Subcellular locationi

    Cytoplasm Curated

    GO - Cellular componenti

    1. cytoplasm Source: UniProtKB-SubCell

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

    3D structure databases

    Select the link destinations:
    PDBe
    RCSB PDB
    PDBj
    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[»]
    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

    MVDGNYSVAS NVMVPMRDGV RLAVDLYRPD ADGPVPVLLV RNPYDKFDVF    50
    AWSTQSTNWL EFVRDGYAVV IQDTRGLFAS EGEFVPHVDD EADAEDTLSW 100
    ILEQAWCDGN VGMFGVSYLG VTQWQAAVSG VGGLKAIAPS MASADLYRAP 150
    WYGPGGALSV EALLGWSALI GTGLITSRSD ARPEDAADFV QLAAILNDVA 200
    GAASVTPLAE QPLLGRLIPW VIDQVVDHPD NDESWQSISL FERLGGLATP 250
    ALITAGWYDG FVGESLRTFV AVKDNADARL VVGPWSHSNL TGRNADRKFG 300
    IAATYPIQEA TTMHKAFFDR HLRGETDALA GVPKVRLFVM GIDEWRDETD 350
    WPLPDTAYTP FYLGGSGAAN TSTGGGTLST SISGTESADT YLYDPADPVP 400
    SLGGTLLFHN GDNGPADQRP IHDRDDVLCY STEVLTDPVE VTGTVSARLF 450
    VSSSAVDTDF TAKLVDVFPD GRAIALCDGI VRMRYRETLV NPTLIEAGEI 500
    YEVAIDMLAT SNVFLPGHRI MVQVSSSNFP KYDRNSNTGG VIAREQLEEM 550
    CTAVNRIHRG PEHPSHIVLP IIKR 574
    Length:574
    Mass (Da):62,132
    Last modified:October 1, 2000 - v1
    Checksum:i9E35724F586089B7
    GO

    Sequence databases

    Select the link destinations:
    EMBL
    GenBank
    DDBJ
    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:
    EMBL
    GenBank
    DDBJ
    Links Updated
    AF173165 Genomic DNA. Translation: AAF42807.1 .

    3D structure databases

    Select the link destinations:
    PDBe
    RCSB PDB
    PDBj
    Links Updated
    Entry Method Resolution (Å) Chain Positions PDBsum
    1JU3 X-ray 1.58 A 1-574 [» ]
    1JU4 X-ray 1.63 A 1-574 [» ]
    1L7Q X-ray 1.76 A 1-574 [» ]
    1L7R X-ray 1.64 A 1-574 [» ]
    3I2F X-ray 2.50 A 1-574 [» ]
    3I2G X-ray 2.50 A 1-574 [» ]
    3I2H X-ray 1.65 A 1-574 [» ]
    3I2I X-ray 2.14 A 1-574 [» ]
    3I2J X-ray 2.01 A 1-574 [» ]
    3I2K X-ray 1.51 A 1-574 [» ]
    3IDA X-ray 1.60 A 1-574 [» ]
    3PUH X-ray 2.30 A/B 1-574 [» ]
    3PUI X-ray 1.53 A 1-574 [» ]
    ProteinModelPortali Q9L9D7.
    SMRi Q9L9D7. Positions 5-574.
    ModBasei Search...
    MobiDBi Search...

    Protocols and materials databases

    Structural Biology Knowledgebase Search...

    Enzyme and pathway databases

    UniPathwayi UPA00110 .
    BioCyci MetaCyc:MONOMER-15371.
    BRENDAi 3.1.1.1. 5397.

    Miscellaneous databases

    EvolutionaryTracei Q9L9D7.

    Family and domain databases

    Gene3Di 2.60.120.260. 1 hit.
    3.40.50.1820. 2 hits.
    InterProi IPR029058. AB_hydrolase.
    IPR005674. CocE/Ser_esterase.
    IPR008979. Galactose-bd-like.
    IPR000383. Xaa-Pro-like_dom.
    IPR013736. Xaa-Pro_dipept_C.
    [Graphical view ]
    Pfami PF02129. Peptidase_S15. 1 hit.
    PF08530. PepX_C. 1 hit.
    [Graphical view ]
    SMARTi SM00939. PepX_C. 1 hit.
    [Graphical view ]
    SUPFAMi SSF49785. SSF49785. 1 hit.
    SSF53474. SSF53474. 1 hit.
    TIGRFAMsi TIGR00976. /NonD. 1 hit.
    ProtoNeti Search...

    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: October 1, 2014
    This is version 86 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