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Information on EC 3.1.1.84 - cocaine esterase and Organism(s) Rhodococcus sp. and UniProt Accession Q9L9D7

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EC Tree
     3 Hydrolases
         3.1 Acting on ester bonds
             3.1.1 Carboxylic-ester hydrolases
                3.1.1.84 cocaine esterase
IUBMB Comments
Rhodococcus sp. strain MB1 and Pseudomonas maltophilia strain MB11L can utilize cocaine as sole source of carbon and energy [2,3].
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This record set is specific for:
Rhodococcus sp.
UNIPROT: Q9L9D7
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Word Map
The taxonomic range for the selected organisms is: Rhodococcus sp.
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Synonyms
cocaine esterase, cocaine hydrolase, coch1, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
cocaine + H2O = ecgonine methyl ester + benzoate
show the reaction diagram
the entire hydrolysis reaction consists of four reaction steps, including the nucleophilic attack on the carbonyl carbon of benzoyl ester group by the hydroxyl group of Ser117, dissociation of benzoyl ester group, nucleophilic attack on the carbonyl carbon of benzoyl ester group by water, and finally dissociation between the (-)-cocaine benzoyl group and Ser117 of CocE. The third reaction step involving the nucleophilic attack of a water molecule is rate-determining
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SYSTEMATIC NAME
IUBMB Comments
cocaine benzoylhydrolase
Rhodococcus sp. strain MB1 and Pseudomonas maltophilia strain MB11L can utilize cocaine as sole source of carbon and energy [2,3].
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(-)-cocaine + H2O
ecgonine methyl ester + benzoate
show the reaction diagram
cocaethylene + H2O
?
show the reaction diagram
cocaethylene is a more potent cocaine metabolite, observed in patients who concurrently abuse cocaine and alcohol
-
-
?
cocaine + H2O
ecgonine methyl ester + benzoate
show the reaction diagram
cocaine + H2O
ecgonine methyl ester + benzoate
show the reaction diagram
-
-
-
-
?
additional information
?
-
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
(-)-cocaine + H2O
ecgonine methyl ester + benzoate
show the reaction diagram
-
-
-
?
cocaine + H2O
ecgonine methyl ester + benzoate
show the reaction diagram
cocaine + H2O
ecgonine methyl ester + benzoate
show the reaction diagram
-
-
-
-
?
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0016
cocaethylene
pH 7.4, wild-type enzyme
0.00027 - 1.33
cocaine
0.0057 - 0.026
cocaine
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
9.4
cocaethylene
pH 7.4, wild-type enzyme
0.046 - 2502
cocaine
40.1 - 56.6
cocaine
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
702 - 38330
cocaine
2110 - 8990
cocaine
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.8 - 10.5
pH 7.8: about 50% of maximal activity, pH 10.0: about 50% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
Rhodococcus sp. MB1 is capable of utilizing cocaine as a sole source of carbon and nitrogen for growth
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
62128
1 * 62128, calculated from sequence
65000
gel filtration
70000
x * 70000, SDS-PAGE
127000
-
-
65000
-
x * 65000, SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 70000, SDS-PAGE
homodimer
x-ray crystallography
monomer
1 * 62128, calculated from sequence
?
-
x * 65000, SDS-PAGE
homodimer
-
x-ray crystallography
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structures of the S117A and Y44F mutants of cocE
hanging drop vapor diffusion method, using 0.1 M phosphate-citrate (pH 4.2), 1.6 M sodium dihydrogen phosphate, and 0.4 M dipotassium hydrogen phosphate
mutant enzyme L169K/G173Q, hanging drop vapor diffusion method, using 1.7 M ammonium sulfate, 10 mM Tris-HCl, pH 7.3, and 25 mM NaCl
te crystal structure of cocE, solved by multiple anomalous dispersion methods, reveals that cocE is a serine esterase composed of three domains: (1.) a canonical alpha/beta hydrolase fold (2.) an alpha-helical domain that caps the active site and (3.) a jelly-roll-like beta-domain that interacts extensively with the other two domains. The active site is identified within the interface of all three domains by analysis of the crystal structures of transition state analog adduct and product complexes, which are refined at 1.58 A and 1.63 A resolution, respectively
hanging drop vapor diffusion method, using 20% (w/v) PEG 3350, 100 mM 2-(N-morpholino)-ethane sulfonic acid, pH 6.0, and 1 M NaCl
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D259N
mutation results in more than 1500fold decrease in kcat
F261A
mutant catalyzed the hydrolysis of cocaine with a 29fold lower kcat and 15fold higher KM
F408A
mutant has 8fold increased KM and more than 100fold decrease in kcat
G173Q
H287A
mutation results in more than 1500fold decrease in kcat
L169K
the mutation significantly increases the stability of cocaine esterase over that of wild type enzyme (half-life at 37°C is 570 min). The mutant exhibits about 8fold increase in Km for cocaine compared to the wild type enzyme
L169K/G173Q
highly thermostable mutant with a half-life of 2.9 days at 37°C
L407A
mutant has 2fold increased KM and more than 100fold decrease in kcat
L407A/F408A
attempts to express the L407A/F408A double mutant do not result in any soluble protein
LMWP-S-S-T172R/G173Q
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
LMWP-T172R/G173Q
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
Q55E
the mutation within the active site of cocE results in a 2fold improvement in KM, but a 14fold loss of kcat
S117A
mutation results in more than 1500fold decrease in kcat, crystal structures of the S117A and Y44F mutants of cocE. The first urea unfolding transition in the S117A mutant is shifted from 0.5 to 1.3 M urea compared to the wild-type, while the second transition, although broader, has a similar transition point
T172R
T172R/G173Q
T172R/G173Q -LMWP
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
T172R/G173Q -YGRKKRRQRRR
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
T172R/G173Q /G4C/S10C
the mutant shows improved catalytic efficiency against cocaine by about 20%
T172R/G173Q/L169K
the mutant shows poor enzyme kinetics and does not display enhanced stabilization
T172R/G173Q/L196C/I301C
the mutant has not only considerably extended the in vitro half-life at 37°C to more than 100 days, but also significantly improved catalytic efficiency against cocaine by about 150%
W151A
mutant catalyzed the hydrolysis of cocaine with a 78fold lower kcat and 80fold higher KM
W166A
mutant has a 29fold lower kcat, and a 6fold increased KM
Y44F
mutation results in more than 1500fold decrease in kcat, crystal structures of the S117A and Y44F mutants of cocE. The urea unfolding curve of the Y44F mutant is very similar to the wild-type, and has almost identical transition points
YGRKKRRQRRR-T172R/G173Q
the mutant still maintains 52% of its cocaine-hydrolyzing efficiency even after incubation at 37°C for 24 h
G173Q/L169K
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the mutant has a half-life of 370 min and 2.9 days at 37°C
G4C/S10C
-
the mutant shows about 4fold reduced catalytic efficiency compared to the wild type enzyme. The mutant retains almost all activity after 7 days of 37°C treatment
T172R/G173Q
-
the mutant shows about 4fold reduced catalytic efficiency compared to the wild type enzyme. The mutant remains more than 90% active for longer than 40 days at 37°C, representing a more than 4700fold improvement over wild type. PEGylated mutant enzyme retains full in vitro enzymatic activity
additional information
computational-experimental effort yields a CocE variant with a 30-fold increase in plasma half-life both in vitro and in vivo
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
the wild type enzyme has a half-life of 12.2 min at 37°C
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
urea
urea denaturation studies of cocE by fluorescence and circular dichroism show two unfolding transitions (0.5-0.6 M and 3.2-3.7 M urea), with the first transition likely representing pertubation of the active site
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
HisPur cobalt resin column chromatography
recombinant enzyme
Talon metal affinity column chromatography and Q-Sepharose column chromatography
Talon metal chelate affinity column chromatography and Q-Sepharose column chromatography
Q-Sepharose column chromatography and phenyl Sepharose column chromatography
-
Talon metal chelate column chromatography and Q-Sepharose column chromatography
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL-21 Gold (DE3) cells
expressed in Escherichia coli BL21 Star (DE3) cells
expressed in Escherichia coli BL21(DE3) cells
the cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. sequence comparison suggests that cocE encodes a serine esterase
expressed in Escherichia coli B834 (DE3) cells
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expressed in Escherichia coli BL-21 Gold (DE3) cells
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Turner, J.M.; Larsen, N.A.; Basran, A.; Barbas, C.F., 3rd; Bruce, N.C.; Wilson, I.A.; Lerner, R.A.
Biochemical characterization and structural analysis of a highly proficient cocaine esterase
Biochemistry
41
12297-12307
2002
Rhodococcus sp. (Q9L9D7)
Manually annotated by BRENDA team
Larsen, N.A.; Turner, J.M.; Stevens, J.; Rosser, S.J.; Basran, A.; Lerner, R.A.; Bruce, N.C.; Wilson, I.A.
Crystal structure of a bacterial cocaine esterase
Nat. Struct. Biol.
9
17-21
2002
Rhodococcus sp. (Q9L9D7)
Manually annotated by BRENDA team
Ko, M.C.; Narasimhan, D.; Berlin, A.A.; Lukacs, N.W.; Sunahara, R.K.; Woods, J.H.
Effects of cocaine esterase following its repeated administration with cocaine in mice
Drug Alcohol Depend.
101
202-209
2009
Rhodococcus sp. (Q9L9D7)
Manually annotated by BRENDA team
Gao, D.; Narasimhan, D.L.; Macdonald, J.; Brim, R.; Ko, M.C.; Landry, D.W.; Woods, J.H.; Sunahara, R.K.; Zhan, C.G.
Thermostable variants of cocaine esterase for long-time protection against cocaine toxicity
Mol. Pharmacol.
75
318-323
2009
Rhodococcus sp. (Q9L9D7)
Manually annotated by BRENDA team
Bresler, M.M.; Rosser, S.J.; Basran, A.; Bruce, N.C.
Gene cloning and nucleotide sequencing and properties of a cocaine esterase from Rhodococcus sp. strain MB1
Appl. Environ. Microbiol.
66
904-908
2000
Rhodococcus sp. (Q9L9D7), Rhodococcus sp., Rhodococcus sp. MB1 (Q9L9D7)
Manually annotated by BRENDA team
Liu, J.; Hamza, A.; Zhan, C.G.
Fundamental reaction mechanism and free energy profile for (-)-cocaine hydrolysis catalyzed by cocaine esterase
J. Am. Chem. Soc.
131
11964-11975
2003
Rhodococcus sp. (Q9L9D7)
Manually annotated by BRENDA team
Collins, G.T.; Brim, R.L.; Narasimhan, D.; Ko, M.C.; Sunahara, R.K.
Zhan. C.G.; Woods. J.H.: Cocaine esterase prevents cocaine-induced toxicity and the ongoing intravenous self-administration of cocaine in rats
J. Pharmacol. Exp. Ther.
331
445-455
2009
Rhodococcus sp. (Q9L9D7)
Manually annotated by BRENDA team
Brim, R.L.; Nance, M.R.; Youngstrom, D.W.; Narasimhan, D.; Zhan, C.G.; Tesmer, J.J.; Sunahara, R.K.; Woods, J.H.
A thermally stable form of bacterial cocaine esterase: a potential therapeutic agent for treatment of cocaine abuse
Mol. Pharmacol.
77
593-600
2010
Rhodococcus sp. (Q9L9D7), Rhodococcus sp. MB1 (Q9L9D7)
Manually annotated by BRENDA team
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.
Subunit stabilization and pegylation of cocaine esterase improves in vivo residence time
Mol. Pharmacol.
80
1056-1065
2011
Rhodococcus sp.
Manually annotated by BRENDA team
Narasimhan, D.; Nance, M.; Gao, D.; Ko, M.; MacDonald, J.; Tamburi, P.; Yoon, D.; Landry, D.; Woods, J.; Zhan, C.; Tesmer, J.; Sunahara, R.
Structural analysis of thermostabilizing mutations of cocaine esterase
Protein Eng.
23
537-547
2010
Rhodococcus sp. (Q9L9D7), Rhodococcus sp. MB1 (Q9L9D7)
Manually annotated by BRENDA team
Fang, L.; Chow, K.M.; Hou, S.; Xue, L.; Chen, X.; Rodgers, D.W.; Zheng, F.; Zhan, C.G.
Rational design, preparation, and characterization of a therapeutic enzyme mutant with improved stability and function for cocaine detoxification
ACS Chem. Biol.
9
1764-1772
2014
Rhodococcus sp. (Q9L9D7), Rhodococcus sp. MB1 Bresler (Q9L9D7)
Manually annotated by BRENDA team
Brim, R.; Noon, K.; Collins, G.; Stein, A.; Nichols, J.; Narasimhan, D.; Ko, M.; Woods, J.; Sunahara, R.
The fate of bacterial cocaine esterase (CocE): An in vivo study of CocE-mediated cocaine hydrolysis, CocE pharmacokinetics, and CocE elimination
J. Pharmacol. Exp. Ther.
340
83-95
2012
Rhodococcus sp.
Manually annotated by BRENDA team
Lee, T.Y.; Park, Y.S.; Garcia, G.A.; Sunahara, R.K.; Woods, J.H.; Yang, V.C.
Cell permeable cocaine esterases constructed by chemical conjugation and genetic recombination
Mol. Pharm.
9
1361-1373
2012
Rhodococcus sp. (Q9L9D7), Rhodococcus sp. MB1 (Q9L9D7)
Manually annotated by BRENDA team