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The taxonomic range for the selected organisms is: Rhodococcus sp.
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
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(-)-cocaine + H2O
ecgonine methyl ester + benzoate
cocaethylene + H2O
?
cocaethylene is a more potent cocaine metabolite, observed in patients who concurrently abuse cocaine and alcohol
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cocaine + H2O
ecgonine methyl ester + benzoate
cocaine + H2O
ecgonine methyl ester + benzoate
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-
-
?
additional information
?
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(-)-cocaine + H2O
ecgonine methyl ester + benzoate
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-
?
(-)-cocaine + H2O
ecgonine methyl ester + benzoate
(-)-cocaine is the naturally occurring enantiomer. 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
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cocaine + H2O
ecgonine methyl ester + benzoate
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cocaine + H2O
ecgonine methyl ester + benzoate
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cocaine + H2O
ecgonine methyl ester + benzoate
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cocaine + H2O
ecgonine methyl ester + benzoate
CocE is the most efficient native enzyme for metabolizing the naturally occurring cocaine
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cocaine + H2O
ecgonine methyl ester + benzoate
Rhodococcus sp. MB1 is capable of utilizing cocaine as a sole source of carbon and nitrogen for growth. The organism lives in the rhizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca. The cocaine esterase initiates degradation of cocaine, which is hydrolyzed to ecgonine methyl ester and benzoate. Both of these esterolytic products are further metabolized by Rhodococcus sp. strain MB1. This activity is inducible in Rhodococcus sp. strain MB1, since no cocaine esterase activity is observed in cells grown on 15 mM succinate as a sole source of carbon
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cocaine + H2O
ecgonine methyl ester + benzoate
the bacterial cocaine esterase, cocE, hydrolyzes cocaine faster than any other reported cocaine esterase
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additional information
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the cocaine esterase displays low levels of activity with 20 mM atropine, a structurally related tropane alkaloid
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additional information
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the cocaine esterase displays low levels of activity with 20 mM atropine, a structurally related tropane alkaloid
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0.0016
cocaethylene
pH 7.4, wild-type enzyme
0.00027
cocaine
pH 7.4, mutant enzyme Q55E
0.00064
cocaine
pH 7.4, wild-type enzyme
0.00075
cocaine
pH 7.4, mutant enzyme Q55A
0.0012
cocaine
pH 7.4, mutant enzyme L407A
0.0015
cocaine
mutant enzyme T172R/G173Q/L196C/I301C, at 37°C, pH not specified in the publication
0.0021
cocaine
mutant enzyme T172R/G173Q/G4C/S10C, at 37°C, pH not specified in the publication
0.0029
cocaine
mutant enzyme T172R/G173Q, at 37°C, pH not specified in the publication
0.0036
cocaine
pH 7.4, mutant enzyme W166A
0.0037
cocaine
mutant enzyme G173Q, at pH 7.4 and 37°C
0.0051
cocaine
pH 7.4, mutant enzyme F408A
0.0057
cocaine
wild type enzyme, at pH 7.4 and 37°C
0.0072
cocaine
wild type enzyme, without preincubation, at pH 7.4 and 37°C
0.0073
cocaine
wild type enzyme, after 24 h preincubation at 37°C, at pH 7.4 and 37°C
0.0096
cocaine
pH 7.4, mutant enzyme F261A
0.015
cocaine
ph 7.4, 37°C, mutant enzyme G173Q
0.017
cocaine
mutant enzyme T172R, at pH 7.4 and 37°C
0.017
cocaine
mutant enzyme T172R/G13Q, at pH 7.4 and 37°C
0.021
cocaine
pH 7.4, 37°C, wild-type enzyme
0.0229
cocaine
mutant enzyme L169K/G173Q, after 24 h preincubation at 37°C, at pH 7.4 and 37°C
0.024
cocaine
ph 7.4, 37°C, mutant enzyme T172R
0.024
cocaine
ph 7.4, 37°C, mutant enzyme T172R/G173Q
0.0288
cocaine
wild type enzyme, at pH 7.4 and 37°C
0.0303
cocaine
mutant enzyme L169K/G173Q, without preincubation, at pH 7.4 and 37°C
0.0305
cocaine
mutant enzyme LMWP-S-S-T172R/G173Q, at pH 7.4 and 37°C
0.0306
cocaine
mutant enzyme T172R/G173Q -YGRKKRRQRRR, at pH 7.4 and 37°C
0.0396
cocaine
mutant enzyme YGRKKRRQRRR-T172R/G173Q, at pH 7.4 and 37°C
0.0415
cocaine
mutant enzyme T172R/G173Q -LMWP, at pH 7.4 and 37°C
0.044
cocaine
mutant enzyme L169K, at pH 7.4 and 37°C
0.046
cocaine
pH 7.4, mutant enzyme S117C
0.049
cocaine
mutant enzyme LMWP-T172R/G173Q, at pH 7.4 and 37°C
0.051
cocaine
pH 7.4, mutant enzyme W151A
1.33
cocaine
pH 7.5, 30°C
0.0057
cocaine
-
wild type enzyme, pH not specified in the publication, 37°C
0.019
cocaine
-
pegylated mutant enzyme T172R/G173Q, pH not specified in the publication, 37°C
0.021
cocaine
-
mutant enzyme G4C/S10C, pH not specified in the publication, 37°C
0.026
cocaine
-
mutant enzyme T172R/G173Q, pH not specified in the publication, 37°C
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9.4
cocaethylene
pH 7.4, wild-type enzyme
0.046
cocaine
pH 7.4, mutant enzyme S117C
0.057
cocaine
pH 7.4, mutant enzyme F408A
0.067
cocaine
pH 7.4, mutant enzyme L407A
0.1
cocaine
pH 7.4, mutant enzyme W151A
0.27
cocaine
pH 7.4, mutant enzyme F261A
0.27
cocaine
pH 7.4, mutant enzyme W166A
0.55
cocaine
pH 7.4, mutant enzyme Q55E
1.7
cocaine
pH 7.4, mutant enzyme Q55A
7.8
cocaine
pH 7.4, wild-type enzyme
31.45
cocaine
mutant enzyme T172R/G173Q -YGRKKRRQRRR, at pH 7.4 and 37°C
32.62
cocaine
mutant enzyme YGRKKRRQRRR-T172R/G173Q, at pH 7.4 and 37°C
34.42
cocaine
mutant enzyme LMWP-T172R/G173Q, at pH 7.4 and 37°C
35.17
cocaine
mutant enzyme T172R/G173Q -LMWP, at pH 7.4 and 37°C
36.1
cocaine
mutant enzyme G173Q, at pH 7.4 and 37°C
39
cocaine
mutant enzyme T172R/G173Q/G4C/S10C, at 37°C, pH not specified in the publication
39.17
cocaine
mutant enzyme LMWP-S-S-T172R/G173Q, at pH 7.4 and 37°C
43.3
cocaine
mutant enzyme T172R/G173Q, at 37°C, pH not specified in the publication
44.85
cocaine
wild type enzyme, at pH 7.4 and 37°C
50.9
cocaine
mutant enzyme T172R, at pH 7.4 and 37°C
51.4
cocaine
wild type enzyme, at pH 7.4 and 37°C
53.4
cocaine
mutant enzyme T172R/G13Q, at pH 7.4 and 37°C
57.7
cocaine
mutant enzyme T172R/G173Q/L196C/I301C, at 37°C, pH not specified in the publication
80.1
cocaine
mutant enzyme L169K, at pH 7.4 and 37°C
2247
cocaine
ph 7.4, 37°C, mutant enzyme T172R/G173Q
2323
cocaine
pH 7.4, 37°C, wild-type enzyme
2384
cocaine
ph 7.4, 37°C, mutant enzyme G173Q
2502
cocaine
ph 7.4, 37°C, mutant enzyme T172R
40.1
cocaine
-
pegylated mutant enzyme T172R/G173Q, pH not specified in the publication, 37°C
49.9
cocaine
-
mutant enzyme G4C/S10C, pH not specified in the publication, 37°C
51.4
cocaine
-
wild type enzyme, pH not specified in the publication, 37°C
56.6
cocaine
-
mutant enzyme T172R/G173Q, pH not specified in the publication, 37°C
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702
cocaine
mutant enzyme LMWP-T172R/G173Q, at pH 7.4 and 37°C
823
cocaine
mutant enzyme YGRKKRRQRRR-T172R/G173Q, at pH 7.4 and 37°C
847
cocaine
mutant enzyme T172R/G173Q -LMWP, at pH 7.4 and 37°C
1028
cocaine
mutant enzyme T172R/G173Q -YGRKKRRQRRR, at pH 7.4 and 37°C
1283
cocaine
mutant enzyme LMWP-S-S-T172R/G173Q, at pH 7.4 and 37°C
1560
cocaine
wild type enzyme, at pH 7.4 and 37°C
1840
cocaine
mutant enzyme L169K, at pH 7.4 and 37°C
3060
cocaine
mutant enzyme T172R, at pH 7.4 and 37°C
3180
cocaine
mutant enzyme T172R/G13Q, at pH 7.4 and 37°C
8990
cocaine
wild type enzyme, at pH 7.4 and 37°C
9880
cocaine
mutant enzyme G173Q, at pH 7.4 and 37°C
15330
cocaine
mutant enzyme T172R/G173Q, at 37°C, pH not specified in the publication
18330
cocaine
mutant enzyme T172R/G173Q/G4C/S10C, at 37°C, pH not specified in the publication
38330
cocaine
mutant enzyme T172R/G173Q/L196C/I301C, at 37°C, pH not specified in the publication
2110
cocaine
-
pegylated mutant enzyme T172R/G173Q, pH not specified in the publication, 37°C
2120
cocaine
-
mutant enzyme T172R/G173Q, pH not specified in the publication, 37°C
2380
cocaine
-
mutant enzyme G4C/S10C, pH not specified in the publication, 37°C
8990
cocaine
-
wild type enzyme, pH not specified in the publication, 37°C
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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
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/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
-
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
G173Q
kcat and Km-value for cocaine is similar to wild-type value, half-life is increased 7fold compared to wild-type enzyme
G173Q
the mutant does not have any deleterious effects on the catalytic efficiency
T172R
kcat and Km-value for cocaine is similar to wild-type value, half-life is increased 7fold compared to wild-type enzyme
T172R
the mutants shows about wild type thermal stability and decreased catalytic efficiency for cocaine
T172R/G173Q
kcat and Km-value for cocaine is similar to wild-type value, half-life is increased 30fold compared to wild-type enzyme
T172R/G173Q
mutant enzyme with increased half-life
T172R/G173Q
the mutation extends half-life at 37°C up to 370 min (30fold improvement compared to the wild type stability) and leads to about 3fold decrease of catalytic efficiency for cocaine
T172R/G173Q
the mutant has an improved in vitro half-life of about 6 h at 37°C
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medicine
a series of experiments demonstrates the capacity of the longer acting mutant, CocE T172R/G173Q, to provide a long-lasting protection against cocaine-induced convulsion and lethality, and a dose-dependent and selective inhibition the ongoing self-administration of cocaine in rats. Potential usefulness of a suitable, stable, and long-acting form of CocE as a pharmacotherapy for cocaine abuse in humans
medicine
because of the high catalytic proficiency of cocE, it is an attractive candidate for novel protein-based therapies for cocaine overdose
medicine
CocE produces robust protection and reversal of cocaine toxicity and provides in vivo evidence for the therapeutic potential of CocE in the treatment of acute cocaine toxicity. Repeated use of CocE may gradually reduce the effectiveness of CocE as a protection or rescue treatment due to the production of anti-CocE antibodies
medicine
enhancing cocaine metabolism by administration of cocaine esterase (CocE) is a promising treatment strategy for cocaine overdose and addiction, because CocE is the most efficient native enzyme for metabolizing the naturally occurring cocaine. A major obstacle to the clinical application of CocE is the thermoinstability of native CocE with a half-life of only a few min at physiological temperature. Computational-experimental effort yields a CocE variant with a 30fold increase in plasma half-life both in vitro and in vivo
medicine
the high catalytic proficiency, lack of observable product inhibition, and ability to hydrolyze both cocaine and cocaethylene make cocE an attractive candidate for rapid cocaine detoxification in an emergency setting
medicine
cocaine esterase is used to accelerate the removal of systemic cocaine and to prevent cocaine-induced lethality
medicine
the wild type cocaine esterase cannot be used as a pharmacotherapy for cocaine abuse because of its 13.7-min half-life at 37°C
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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)
brenda
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)
brenda
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)
brenda
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)
brenda
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)
brenda
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)
brenda
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)
brenda
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)
brenda
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.
brenda
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)
brenda
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)
brenda
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.
brenda
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)
brenda