Q9L9D7 (COCE_RHOSM) Reviewed, UniProtKB/Swiss-Prot

Names and origin

Protein names

Recommended name:
Cocaine esterase
EC=3.1.1.84

Gene names

Name:

cocE

Organism

Rhodococcus sp. (strain MB1 Bresler)

Taxonomic identifier

104109 [NCBI]

Taxonomic lineage

cellular organisms › Bacteria › Actinobacteria › Actinobacteria › Actinobacteridae › Actinomycetales › Corynebacterineae › Nocardiaceae › Rhodococcus

Protein attributes

Sequence length	574 AA.
Sequence status	Complete.
Protein existence	Evidence 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 component	Cytoplasm
Molecular function	Hydrolase Serine esterase
Technical term	3D-structure Direct protein sequencing
Gene Ontology (GO)
Biological_process	cocaine catabolic process Inferred from electronic annotation. Source: UniProtKB-UniPathway proteolysis Inferred from electronic annotation. Source: InterPro
Cellular_component	cytoplasm Inferred from electronic annotation. Source: UniProtKB-SubCell
Molecular_function	aminopeptidase activity Inferred from electronic annotation. Source: InterPro carboxylesterase activity Inferred from electronic annotation. Source: UniProtKB-KW dipeptidyl-peptidase activity Inferred from electronic annotation. Source: InterPro
Complete GO annotation...

Sequence annotation (Features)

Feature key

Position(s)

Length

Description

Graphical view

Feature identifier

Molecule processing

Chain

1 – 574

574

Cocaine esterase

PRO_0000090000

Regions

Region

1 – 144

144

1A

Region

145 – 240

96

2

Region

241 – 354

114

1B

Region

355 – 574

220

3

Sites

Active site

117

1

Acyl-ester intermediate Ref.3

Active site

259

1

Charge relay system Ref.3

Active site

287

1

Charge relay system Ref.3

Binding site

44

1

Substrate

Binding site

118

1

Substrate; via amide nitrogen

Site

44

1

Probably involved in activating the substrate carbonyl and the acyl enzyme for hydrolysis

Experimental info

Mutagenesis

44

1

Y → F: Loss of activity. Has no protective effects against cocaine-induced convulsions and lethality in rat. Ref.2 Ref.4

Mutagenesis

55

1

Q → A or E: Decrease in activity. Ref.4

Mutagenesis

117

1

S → A: Loss of activity. Has no protective effects against cocaine-induced convulsions and lethality in rat. Ref.2 Ref.4

Mutagenesis

117

1

S → C: Great decrease in activity. Ref.2 Ref.4

Mutagenesis

151

1

W → A: Decrease in activity. Ref.4

Mutagenesis

166

1

W → A: Decrease in activity. Ref.4

Mutagenesis

169

1

L → 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

Mutagenesis

172

1

T → 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

Mutagenesis

173

1

G → 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

Mutagenesis

259

1

D → N: Loss of activity. Ref.4

Mutagenesis

261

1

F → A: Decrease in activity. Ref.4

Mutagenesis

287

1

H → A: Loss of activity. Ref.4

Mutagenesis

407

1

L → A: Decrease in activity. Ref.4

Mutagenesis

408

1

F → A: Decrease in activity. Ref.4

Secondary structure

1

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574

Helix Strand Turn

Details...

Beta strand

6 – 15

10

Beta strand

21 – 29

9

Beta strand

35 – 44

10

Helix

49 – 53

5

Turn

54 – 56

3

Helix

60 – 64

5

Beta strand

68 – 73

6

Turn

86 – 89

4

Helix

90 – 103

14

Beta strand

107 – 113

7

Helix

118 – 127

10

Beta strand

134 – 137

4

Beta strand

139 – 141

3

Helix

147 – 151

5

Helix

160 – 177

18

Beta strand

178 – 180

3

Helix

185 – 196

12

Helix

199 – 203

5

Beta strand

205 – 207

3

Helix

212 – 217

6

Helix

220 – 223

4

Turn

224 – 227

4

Helix

233 – 236

4

Helix

241 – 244

4

Beta strand

251 – 258

8

Helix

262 – 272

11

Turn

273 – 275

3

Beta strand

278 – 286

9

Beta strand

291 – 294

4

Helix

301 – 303

3

Helix

307 – 322

16

Turn

326 – 331

6

Beta strand

334 – 340

7

Turn

341 – 343

3

Beta strand

344 – 352

9

Beta strand

357 – 364

8

Beta strand

377 – 381

5

Beta strand

387 – 393

7

Beta strand

407 – 410

4

Helix

419 – 421

3

Beta strand

428 – 431

4

Beta strand

439 – 457

19

Beta strand

459 – 467

9

Beta strand

473 – 482

10

Helix

483 – 485

3

Beta strand

489 – 491

3

Beta strand

501 – 514

14

Beta strand

519 – 526

8

Beta strand

537 – 540

4

Helix

542 – 544

3

Helix

547 – 549

3

Beta strand

553 – 563

11

Beta strand

566 – 572

7

Sequences

	Sequence		Length	Mass (Da)
	Q9L9D7 [UniParc]. Last modified October 1, 2000. Version 1. Checksum: 9E35724F586089B7	FASTA	574	62,132
	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

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

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	[»]

ProteinModelPortal

Q9L9D7.

SMR

Q9L9D7. Positions 5-574.

ModBase

Search...

Protocols and materials databases

StructuralBiologyKnowledgebase

Search...

Enzyme and pathway databases

BioCyc

MetaCyc:MONOMER-15371.

BRENDA

3.1.1.1. 5397.

UniPathway

UPA00110.

Family and domain databases

InterPro

IPR005674. CocE_NonD_hydro.
IPR008979. Galactose-bd-like.
IPR013736. Pept_S15/CocE/NonD_C.
IPR000383. X-Pro-like_dom.
[Graphical view]

Pfam

PF02129. Peptidase_S15. 1 hit.
PF08530. PepX_C. 1 hit.
[Graphical view]

SMART

SM00939. PepX_C. 1 hit.
[Graphical view]

SUPFAM

SSF49785. Gal_bind_like. 1 hit.

TIGRFAMs

TIGR00976. /NonD. 1 hit.

ProtoNet

Search...

Other

EvolutionaryTrace

Q9L9D7.

Entry information

Entry name

COCE_RHOSM

Accession

Primary (citable) accession number: Q9L9D7

Entry history

Integrated into UniProtKB/Swiss-Prot:	January 31, 2002
Last sequence update:	October 1, 2000
Last modified:	April 3, 2013
This is version 78 of the entry and version 1 of the sequence. [Complete history]

Entry status

Reviewed (UniProtKB/Swiss-Prot)

Annotation program

Prokaryotic Protein Annotation Program