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Q9L9D7

- COCE_RHOSM

UniProt

Q9L9D7 - COCE_RHOSM

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Protein

Cocaine esterase

Gene

cocE

Organism
Rhodococcus sp. (strain MB1 Bresler)
Status
Reviewed - 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

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. carboxylic ester hydrolase activity Source: UniProtKB-KW
  2. dipeptidyl-peptidase activity Source: InterPro

GO - Biological processi

  1. cocaine catabolic process Source: UniProtKB-UniPathway
Complete GO annotation...

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-KW
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
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 [» ]
4P08 X-ray 2.34 A 4-574 [» ]
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: November 26, 2014
This is version 88 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