Gene_locus Report for: rhosm-cocE

Computational enzyme design exhibits excellent performance for identifying potential scaffolds from structural databases and creating new enzymatic catalysts from naught. Using the active site-matching algorithm ProdaMatch, we identified a new scaffold cocaine esterase from Rhodococcus sp. that showed modest activity (kcat/Km = 0.018 M-1 s-1) towards the hydrolysis of beta-lactam antibiotic cephradine. The identified cocaine esterase scaffold afforded low sequence identity (<30%) with the known beta-lactam synthases, such as penicillin G acylase or alpha-amino acid ester hydrolase, and was able to catalyze the condensation reaction between d-dihydrophenylglycine methyl ester and 7-aminodesacetoxycephalosporanic acid to produce cephradine via a kinetically controlled synthesis. By virtue of the computational enzyme design protocol, hundreds of sequences were predicted in the cocaine esterase scaffold to promote the catalytic activity towards the hydrolytic reaction of cephradine. Moreover, a single mutant (F261T) was experimentally confirmed to have improved the catalytic efficiency by ten times (kcat/Km = 0.193 M-1 s-1), indicating that the novel scaffold cocaine esterase may be potentially redesigned to become an industrially useful cephradine synthase.

A strain of Rhodococcus designated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca. A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized by Rhodococcus sp. strain MB1. The structural gene encoding a cocaine esterase, designated cocE, was cloned from Rhodococcus sp. strain MB1 genomic libraries by screening recombinant strains of Rhodococcus erythropolis CW25 for growth on cocaine. The nucleotide sequence of cocE corresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. The cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with an M(r) of approximately 65,000. The apparent K(m) of the enzyme (mean +/- standard deviation) for cocaine was measured as 1.33 +/- 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine.

Computational enzyme design exhibits excellent performance for identifying potential scaffolds from structural databases and creating new enzymatic catalysts from naught. Using the active site-matching algorithm ProdaMatch, we identified a new scaffold cocaine esterase from Rhodococcus sp. that showed modest activity (kcat/Km = 0.018 M-1 s-1) towards the hydrolysis of beta-lactam antibiotic cephradine. The identified cocaine esterase scaffold afforded low sequence identity (<30%) with the known beta-lactam synthases, such as penicillin G acylase or alpha-amino acid ester hydrolase, and was able to catalyze the condensation reaction between d-dihydrophenylglycine methyl ester and 7-aminodesacetoxycephalosporanic acid to produce cephradine via a kinetically controlled synthesis. By virtue of the computational enzyme design protocol, hundreds of sequences were predicted in the cocaine esterase scaffold to promote the catalytic activity towards the hydrolytic reaction of cephradine. Moreover, a single mutant (F261T) was experimentally confirmed to have improved the catalytic efficiency by ten times (kcat/Km = 0.193 M-1 s-1), indicating that the novel scaffold cocaine esterase may be potentially redesigned to become an industrially useful cephradine synthase.

Cocaine esterase (CocE) is known as the most efficient natural enzyme for cocaine hydrolysis. The major obstacle to the clinical application of wild-type CocE is the thermoinstability with a half-life of only approximately 12 min at 37 degrees C. The previously designed T172R/G173Q mutant (denoted as enzyme E172-173) with an improved in vitro half-life of approximately 6 h at 37 degrees C is currently in clinical trial Phase II for cocaine overdose treatment. Through molecular modeling and dynamics simulation, we designed and characterized a promising new mutant of E172-173 with extra L196C/I301C mutations (denoted as enzyme E196-301) to produce cross-subunit disulfide bonds that stabilize the dimer structure. The cross-subunit disulfide bonds were confirmed by X-ray diffraction. The designed L196C/I301C mutations have not only considerably extended the in vitro half-life at 37 degrees C to >100 days, but also significantly improved the catalytic efficiency against cocaine by approximately 150%. In addition, the thermostable E196-301 can be PEGylated to significantly prolong the residence time in mice. The PEGylated E196-301 can fully protect mice from a lethal dose of cocaine (180 mg/kg, LD100) for at least 3 days, with an average protection time of approximately 94h. This is the longest in vivo protection of mice from the lethal dose of cocaine demonstrated within all studies using an exogenous enzyme reported so far. Hence, E196-301 may be developed to become a more valuable therapeutic enzyme for cocaine abuse treatment, and it demonstrates that a general design strategy and protocol to simultaneously improve both the stability and function are feasible for rational protein drug design.

No small-molecule therapeutic is available to treat cocaine addiction, but enzyme-based therapy to accelerate cocaine hydrolysis in serum has gained momentum. Bacterial cocaine esterase (CocE) is the fastest known native enzyme that hydrolyzes cocaine. However, its lability at 37 degrees C has limited its therapeutic potential. Cross-linking subunits through disulfide bridging is commonly used to stabilize multimeric enzymes. Herein we use structural methods to guide the introduction of two cysteine residues within dimer interface of CocE to facilitate intermolecular disulfide bond formation. The disulfide-crosslinked enzyme displays improved thermostability, particularly when combined with previously described mutations that enhance stability (T172R-G173Q). The newly modified enzyme yielded an extremely stable form of CocE (CCRQ-CocE) that retained greater than 90% of its activity after 41 days at 37 degrees C, representing an improvement of more than 4700-fold over the wild-type enzyme. CCRQ-CocE could also be modified by polyethylene glycol (PEG) polymers, which improved its in vivo residence time from 24 to 72 h, as measured by a cocaine lethality assay, by self-administration in rodents, and by measurement of inhibition of cocaine-induced cardiovascular effects in rhesus monkeys. PEG-CCRQ elicited negligible immune response in rodents. Subunit stabilization and PEGylation has thus produced a potential protein therapeutic with markedly higher stability both in vitro and in vivo.

Rhodococcal cocaine esterase (CocE) is an attractive potential treatment for both cocaine overdose and cocaine addiction. CocE directly degrades cocaine into inactive products, whereas traditional small-molecule approaches require blockade of the inhibitory action of cocaine on a diverse array of monoamine transporters and ion channels. The usefulness of wild-type (wt) cocaine esterase is hampered by its inactivation at 37 degrees C. Herein, we characterize the most thermostable form of this enzyme to date, CocE-L169K/G173Q. In vitro kinetic analyses reveal that CocE-L169K/G173Q displays a half-life of 2.9 days at 37 degrees C, which represents a 340-fold improvement over wt and is 15-fold greater than previously reported mutants. Crystallographic analyses of CocE-L169K/G173Q, determined at 1.6-A resolution, suggest that stabilization involves enhanced domain-domain interactions involving van der Waals interactions and hydrogen bonding. In vivo rodent studies reveal that intravenous pretreatment with CocE-L169K/G173Q in mice provides protection from cocaine-induced lethality for longer time periods before cocaine administration than wt CocE. Furthermore, intravenous administration (pretreatment) of CocE-L169K/G173Q prevents self-administration of cocaine in a time-dependent manner. Termination of the in vivo effects of CoCE seems to be dependent on, but not proportional to, its clearance from plasma as its half-life is approximately 2.3 h and similar to that of wt CocE (2.2 h). Taken together these data suggest that CocE-L169K/G173Q possesses many of the properties of a biological therapeutic for treating cocaine abuse but requires additional development to improve its serum half-life.

Cocaine is considered to be the most addictive of all substances of abuse and mediates its effects by inhibiting monoamine transporters, primarily the dopamine transporters. There are currently no small molecules that can be used to combat its toxic and addictive properties, in part because of the difficulty of developing compounds that inhibit cocaine binding without having intrinsic effects on dopamine transport. Most of the effective cocaine inhibitors also display addictive properties. We have recently reported the use of cocaine esterase (CocE) to accelerate the removal of systemic cocaine and to prevent cocaine-induced lethality. However, wild-type CocE is relatively unstable at physiological temperatures (tau(1/2) approximately 13 min at 37 degrees C), presenting challenges for its development as a viable therapeutic agent. We applied computational approaches to predict mutations to stabilize CocE and showed that several of these have increased stability both in vitro and in vivo, with the most efficacious mutant (T172R/G173Q) extending half-life up to 370 min. Here we present novel X-ray crystallographic data on these mutants that provide a plausible model for the observed enhanced stability. We also more extensively characterize the previously reported variants and report on a new stabilizing mutant, L169K. The improved stability of these engineered CocE enzymes will have a profound influence on the use of this protein to combat cocaine-induced toxicity and addiction in humans.

The bacterial cocaine esterase, cocE, hydrolyzes cocaine faster than any other reported cocaine esterase. Hydrolysis of the cocaine benzoyl ester follows Michaelis-Menten kinetics with k(cat) = 7.8 s(-1) and K(M) = 640 nM. A similar rate is observed for hydrolysis of cocaethylene, a more potent cocaine metabolite that has been observed in patients who concurrently abuse cocaine and alcohol. 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. Recently, we determined the crystal structure of this enzyme, and showed that it is a serine carboxylesterase, with a catalytic triad formed by S117, H287, and D259 within a hydrophobic active site, and an oxyanion hole formed by the backbone amide of Y118 and the Y44 hydroxyl. The only enzyme previously known to use a Tyr side chain to form the oxyanion hole is prolyl oligopeptidase, but the Y44F mutation of cocE has a more deleterious effect on the specificity rate constant (k(cat)/K(M)) than the analogous Y473F mutation of prolyl oligopeptidase. Kinetic studies on a series of cocE mutants both validate the proposed mechanism, and reveal the relative contributions of active site residues toward substrate recognition and catalysis. Inspired by the anionic binding pocket of the cocaine binding antibody GNC92H2, we found that a Q55E mutation within the active site of cocE results in a modest (2-fold) improvement in K(M), but a 14-fold loss of k(cat). The pH rate profile of cocE was fit to the ionization of two groups (pK(a1) = 7.7; pK(a2) = 10.4) that likely represent titration of H287 and Y44, respectively. We also describe the crystal structures of both S117A and Y44F mutants of cocE. Finally, 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.

A strain of Rhodococcus designated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca. A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized by Rhodococcus sp. strain MB1. The structural gene encoding a cocaine esterase, designated cocE, was cloned from Rhodococcus sp. strain MB1 genomic libraries by screening recombinant strains of Rhodococcus erythropolis CW25 for growth on cocaine. The nucleotide sequence of cocE corresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. The cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with an M(r) of approximately 65,000. The apparent K(m) of the enzyme (mean +/- standard deviation) for cocaine was measured as 1.33 +/- 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine.