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Q9L9D7 (COCE_RHOSM) Reviewed, UniProtKB/Swiss-Prot

Last modified February 19, 2014. Version 82. Feed History...

Clusters with 100%, 90%, 50% identity | Documents (4) | Third-party data text xml rdf/xml gff fasta
to top of pageNames·Attributes·General annotation·Ontologies·Sequence annotation·Sequences·References·Web links·Cross-refs·Entry info·DocumentsCustomize order

Names and origin

Protein namesRecommended name:
Cocaine esterase

EC=3.1.1.84
Gene names
Name:cocE
OrganismRhodococcus sp. (strain MB1 Bresler)
Taxonomic identifier104109 [NCBI]
Taxonomic lineageBacteriaActinobacteriaActinobacteridaeActinomycetalesCorynebacterineaeNocardiaceaeRhodococcus

Protein attributes

Sequence length574 AA.
Sequence statusComplete.
Protein existenceEvidence at protein level

General annotation (Comments)

Function

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. Ref.1 Ref.2 Ref.4

Catalytic activity

Cocaine + H2O = ecgonine methyl ester + benzoate. Ref.1 Ref.4 Ref.5

Pathway

Alkaloid degradation; cocaine degradation.

Subunit structure

Homodimer. The protein aggregates upon heat inactivation. Ref.5

Subcellular location

Cytoplasm Probable.

Induction

Positively induced by cocaine. Ref.1

Domain

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. Ref.3

Biotechnological use

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. Ref.1 Ref.4

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.

Sequence similarities

Belongs to the CocE/NonD hydrolase family.

Biophysicochemical properties

Kinetic parameters:

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

KM=0.64 µM for cocaine Ref.4

KM=1.6 µM for cocaethylene

pH dependence:

Optimum pH is 9.0.

Temperature dependence:

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

Ontologies

Keywords
   Cellular componentCytoplasm
   Molecular functionHydrolase
Serine esterase
   Technical term3D-structure
Direct protein sequencing
Gene Ontology (GO)
   Biological_processcocaine catabolic process

Inferred from electronic annotation. Source: UniProtKB-UniPathway

proteolysis

Inferred from electronic annotation. Source: InterPro

   Cellular_componentcytoplasm

Inferred from electronic annotation. Source: UniProtKB-SubCell

   Molecular_functionaminopeptidase activity

Inferred from electronic annotation. Source: InterPro

dipeptidyl-peptidase activity

Inferred from electronic annotation. Source: InterPro

Complete GO annotation...

Sequence annotation (Features)

Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifier

Molecule processing

Chain1 – 574574Cocaine esterase
PRO_0000090000

Regions

Region1 – 1441441A
Region145 – 240962
Region241 – 3541141B
Region355 – 5742203

Sites

Active site1171Acyl-ester intermediate Ref.3
Active site2591Charge relay system Ref.3
Active site2871Charge relay system Ref.3
Binding site441Substrate
Binding site1181Substrate; via amide nitrogen
Site441Probably involved in activating the substrate carbonyl and the acyl enzyme for hydrolysis

Experimental info

Mutagenesis441Y → F: Loss of activity. Has no protective effects against cocaine-induced convulsions and lethality in rat. Ref.2 Ref.4
Mutagenesis551Q → A or E: Decrease in activity. Ref.4
Mutagenesis1171S → A: Loss of activity. Has no protective effects against cocaine-induced convulsions and lethality in rat. Ref.2 Ref.4
Mutagenesis1171S → C: Great decrease in activity. Ref.2 Ref.4
Mutagenesis1511W → A: Decrease in activity. Ref.4
Mutagenesis1661W → A: Decrease in activity. Ref.4
Mutagenesis1691L → K: Displays greatly enhanced stability, with a half-life of 570 minutes at 37 degrees Celsius. Exhibits 4.5-fold reduction in catalytic efficiency. Ref.5
Mutagenesis1721T → 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. Ref.5
Mutagenesis1731G → 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. Ref.5
Mutagenesis2591D → N: Loss of activity. Ref.4
Mutagenesis2611F → A: Decrease in activity. Ref.4
Mutagenesis2871H → A: Loss of activity. Ref.4
Mutagenesis4071L → A: Decrease in activity. Ref.4
Mutagenesis4081F → A: Decrease in activity. Ref.4

Secondary structure

................................................................................................... 574
Helix Strand Turn

Details...

Sequences

Sequence LengthMass (Da)Tools
Q9L9D7 [UniParc].

Last modified October 1, 2000. Version 1.
Checksum: 9E35724F586089B7

FASTA57462,132
        10         20         30         40         50         60 
MVDGNYSVAS NVMVPMRDGV RLAVDLYRPD ADGPVPVLLV RNPYDKFDVF AWSTQSTNWL 

        70         80         90        100        110        120 
EFVRDGYAVV IQDTRGLFAS EGEFVPHVDD EADAEDTLSW ILEQAWCDGN VGMFGVSYLG 

       130        140        150        160        170        180 
VTQWQAAVSG VGGLKAIAPS MASADLYRAP WYGPGGALSV EALLGWSALI GTGLITSRSD 

       190        200        210        220        230        240 
ARPEDAADFV QLAAILNDVA GAASVTPLAE QPLLGRLIPW VIDQVVDHPD NDESWQSISL 

       250        260        270        280        290        300 
FERLGGLATP ALITAGWYDG FVGESLRTFV AVKDNADARL VVGPWSHSNL TGRNADRKFG 

       310        320        330        340        350        360 
IAATYPIQEA TTMHKAFFDR HLRGETDALA GVPKVRLFVM GIDEWRDETD WPLPDTAYTP 

       370        380        390        400        410        420 
FYLGGSGAAN TSTGGGTLST SISGTESADT YLYDPADPVP SLGGTLLFHN GDNGPADQRP 

       430        440        450        460        470        480 
IHDRDDVLCY STEVLTDPVE VTGTVSARLF VSSSAVDTDF TAKLVDVFPD GRAIALCDGI 

       490        500        510        520        530        540 
VRMRYRETLV NPTLIEAGEI YEVAIDMLAT SNVFLPGHRI MVQVSSSNFP KYDRNSNTGG 

       550        560        570 
VIAREQLEEM CTAVNRIHRG PEHPSHIVLP IIKR 

« Hide

References

[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]"Crystal structure of a bacterial cocaine esterase."
Larsen N.A., Turner J.M., Stevens J., Rosser S.J., Basran A., Lerner R.A., Bruce N.C., Wilson I.A.
Nat. Struct. Biol. 9:17-21(2002) [PubMed] [Europe PMC] [Abstract]
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]"Structural analysis of thermostabilizing mutations of cocaine esterase."
Narasimhan D., Nance M.R., Gao D., Ko M.C., Macdonald J., Tamburi P., Yoon D., Landry D.M., Woods J.H., Zhan C.G., Tesmer J.J., Sunahara R.K.
Protein Eng. Des. Sel. 23:537-547(2010) [PubMed] [Europe PMC] [Abstract]
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.
+Additional computationally mapped references.

Web resources

Protein Spotlight

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

Cross-references

Sequence databases

EMBL
GenBank
DDBJ
AF173165 Genomic DNA. Translation: AAF42807.1.

3D structure databases

PDBe
RCSB PDB
PDBj
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[»]
ProteinModelPortalQ9L9D7.
SMRQ9L9D7. Positions 5-574.
ModBaseSearch...
MobiDBSearch...

Protocols and materials databases

StructuralBiologyKnowledgebaseSearch...

Enzyme and pathway databases

BioCycMetaCyc:MONOMER-15371.
BRENDA3.1.1.1. 5397.
UniPathwayUPA00110.

Family and domain databases

Gene3D2.60.120.260. 1 hit.
InterProIPR005674. CocE_NonD_hydro.
IPR008979. Galactose-bd-like.
IPR013736. Pept_S15/CocE/NonD_C.
IPR000383. X-Pro-like_dom.
[Graphical view]
PfamPF02129. Peptidase_S15. 1 hit.
PF08530. PepX_C. 1 hit.
[Graphical view]
SMARTSM00939. PepX_C. 1 hit.
[Graphical view]
SUPFAMSSF49785. SSF49785. 1 hit.
TIGRFAMsTIGR00976. /NonD. 1 hit.
ProtoNetSearch...

Other

EvolutionaryTraceQ9L9D7.

Entry information

Entry nameCOCE_RHOSM
AccessionPrimary (citable) accession number: Q9L9D7
Entry history
Integrated into UniProtKB/Swiss-Prot: January 31, 2002
Last sequence update: October 1, 2000
Last modified: February 19, 2014
This is version 82 of the entry and version 1 of the sequence. [Complete history]
Entry statusReviewed (UniProtKB/Swiss-Prot)
Annotation programProkaryotic Protein Annotation Program

Relevant documents

SIMILARITY comments

Index of protein domains and families

Protein Spotlight

Protein Spotlight articles and cited UniProtKB/Swiss-Prot entries

PDB cross-references

Index of Protein Data Bank (PDB) cross-references

PATHWAY comments

Index of metabolic and biosynthesis pathways