Basran A

References (4)

Title : Crystal structure of a bacterial cocaine esterase -
Author(s) : Larsen NA , Turner JM , Stevens J , Rosser SJ , Basran A , Lerner RA , Bruce NC , Wilson IA
Ref : Nat Struct Biol , 9 :17 , 2002
PubMedID: 11742345
Gene_locus related to this paper: rhosm-cocE

Title : Biochemical characterization and structural analysis of a highly proficient cocaine esterase - Turner_2002_Biochemistry_41_12297
Author(s) : Turner JM , Larsen NA , Basran A , Barbas CF, 3rd , Bruce NC , Wilson IA , Lerner RA
Ref : Biochemistry , 41 :12297 , 2002
Abstract : 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.
ESTHER : Turner_2002_Biochemistry_41_12297
PubMedSearch : Turner_2002_Biochemistry_41_12297
PubMedID: 12369817
Gene_locus related to this paper: rhosm-cocE

Title : Observation of an arsenic adduct in an acetyl esterase crystal structure - Zhu_2003_J.Biol.Chem_278_2008
Author(s) : Zhu X , Larsen NA , Basran A , Bruce NC , Wilson IA
Ref : Journal of Biological Chemistry , 278 :2008 , 2002
Abstract : The crystal structures of an acetyl esterase, HerE, and its complex with an inhibitor dimethylarsinic acid have been determined at 1.30- and 1.45-A resolution, respectively. Although the natural substrate for the enzyme is unknown, HerE hydrolyzes the acetyl groups from heroin to yield morphine and from phenyl acetate to yield phenol. Recently, the activity of the enzyme toward heroin has been exploited to develop a heroin biosensor, which affords higher sensitivity than other currently available detection methods. The crystal structure reveals a single domain with the canonical alpha/beta hydrolase fold with an acyl binding pocket that snugly accommodates the acetyl substituent of the substrate and three backbone amides that form a tripartite oxyanion hole. In addition, a covalent adduct was observed between the active site serine and dimethylarsinic acid, which inhibits the enzyme. This crystal structure provides the first example of an As-containing compound in a serine esterase active site and the first example of covalent modification of serine by arsenic. Thus, the HerE complex reveals the structural basis for the broad scope inhibition of serine hydrolases by As(V)-containing organic compounds.
ESTHER : Zhu_2003_J.Biol.Chem_278_2008
PubMedSearch : Zhu_2003_J.Biol.Chem_278_2008
PubMedID: 12421810
Gene_locus related to this paper: rhosp-hercx

Title : Gene cloning and nucleotide sequencing and properties of a cocaine esterase from Rhodococcus sp. strain MB1 - Bresler_2000_Appl.Environ.Microbiol_66_904
Author(s) : Bresler MM , Rosser SJ , Basran A , Bruce NC
Ref : Applied Environmental Microbiology , 66 :904 , 2000
Abstract : 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.
ESTHER : Bresler_2000_Appl.Environ.Microbiol_66_904
PubMedSearch : Bresler_2000_Appl.Environ.Microbiol_66_904
PubMedID: 10698749
Gene_locus related to this paper: rhosm-cocE