Bioluminescent Assay for Heroin and Its Metabolites

Holt, P.J.A.; Bruce, Neil C.; Lowe, Christopher R.

doi:10.1021/ac951207r

Cited by 18 publications

(13 citation statements)

References 27 publications

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“…The HerE enzyme is the first structure reported of an esterase known to hydrolyze heroin. Hence, the structure provides a framework for improving the specificity and sensitivity of a recently reported heroin biosensor (2). The high bimolecular rate constant for phenyl acetate (approximately 1000-fold higher than 6-acetylmorphine) implies that with engineering of the active site, the specificity constant for heroin could be substantially improved.…”

Section: Discussionmentioning

confidence: 99%

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Observation of an Arsenic Adduct in an Acetyl Esterase Crystal Structure

Zhu¹,

Larsen²,

Basran³

et al. 2003

Journal of Biological Chemistry

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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-Å 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 ␣/␤ 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.Heroin (3,6-diacetylmorphine) is a highly addictive, synthetic derivative of the alkaloid morphine (Fig. 1A). The instability of the 3-acetyl suggests that it can undergo rapid spontaneous hydrolysis to give 6-acetylmorphine. HerE is known to hydrolyze the 6-acetyl of 6-acetylmorphine to yield morphine (k cat ϭ 12.6 Ϯ 0.6 s Ϫ1 , K m ϭ 0.5 Ϯ 0.06 mM) and was originally identified from the Rhodococcus sp. strain H1 by selective growth using heroin as the sole carbon source (1). Identification of this esterase has facilitated development of a coupled enzyme biosensor for heroin detection (Fig. 1A) with higher sensitivity than other currently available technology (2). In brief, hydrolysis of heroin by HerE generates morphine, which is the substrate for NADPH-dependent morphine dehydrogenase. The activity of morphine dehydrogenase is directly monitored by bacterial luciferase, which outputs a bioluminescent signal at 490 nm (Fig. 1A) (2). The higher activity (k cat K m Ϫ1 ) of HerE toward smaller substrates such as phenyl acetate (Fig. 1B) (k cat ϭ 3.0 Ϯ 0.05 s Ϫ1 , K m ϭ 70 Ϯ 8 nM at pH ϭ 6.4) provides a benchmark for engineering the esterase binding pocket to accept bulkier substrates more efficiently. Hence, the crystal structure of this acetyl esterase should provide substantial insights for structure-based design of second generation heroin biosensors with improved sensitivity.In addition, our structure with the inhibitor dimethylarsinic acid (Fig. 1B) demonstrates for the first time the covalent modification of serine by arsenic. Arsenic lies directly below phosphorous in the periodic table and shares some similar properties, but in general, arsenic is more reactive. As(V)-containing organic compounds comprise a known class of broad scope serine esterase/protease inhibitors (3, 4). Usually, arsonic R-As(V)O 3 H 2 and arsin...

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Section: Discussionmentioning

confidence: 99%

“…1A) (2). The higher activity (k cat K m Ϫ1 ) of HerE toward smaller substrates such as phenyl acetate (Fig.…”

mentioning

confidence: 92%

Observation of an Arsenic Adduct in an Acetyl Esterase Crystal Structure

Zhu¹,

Larsen²,

Basran³

et al. 2003

Journal of Biological Chemistry

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“…Finally, the discovery of this soluble cocaine esterase is of particular interest for its use in a potential illicit-drug sensor. Enzymatic detection of illicit heroin has already been described (12). In conjunction with a suitable NADP ϩ -dependent dehydrogenase active against ecgonine methyl ester, the cocaine esterase could enable simultaneous detection of heroin and cocaine, using established bioluminescence technology (12).…”

Section: Vol 66 2000mentioning

confidence: 99%

“…We envisaged that the isolation of microorganisms from the environment capable of metabolizing drugs, such as cocaine and heroin, as carbon sources for growth might provide a good source of enzymes which could be utilized as recognition components in biosensors for the detection of illicit drugs. Previous work in our laboratory has shown that the enzymes initiating heroin metabolism in bacteria could be successfully used in conjunction with bacterial luciferase for the detection of nanogram quantities of heroin (5,12,24). Work has subsequently been directed towards identifying appropriate enzymes active against cocaine.…”

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confidence: 99%

Gene Cloning and Nucleotide Sequencing and Properties of a Cocaine Esterase fromRhodococcussp. Strain MB1

Bresler

Rosser

Basran

et al. 2000

Appl Environ Microbiol

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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.

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“…The first steps in the transformation of morphine and codeine are oxidation of the 6-hydroxyl group to a ketone by a NADP +dependent morphine dehydrogenase (MDH) to give morphinone and codeinone ( Figure 1) [1][2][3], and saturation of the 7,8-double bond by an NADH-dependent morphinone reductase to give hydromorphone and hydrocodone [4][5][6]. Oxidation of morphine by MDH was made the basis of a rapid and highly sensitive assay for opiates in urine [7,8], and the combined MDH-morphinone…”

Section: Introductionmentioning

confidence: 99%

Mechanistic studies of morphine dehydrogenase and stabilization against covalent inactivation

Walker¹,

French²,

Rathbone

et al. 2000

Biochemical Journal

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Morphine dehydrogenase (MDH) of Pseudomonas putida M10 catalyses the NADP(+)-dependent oxidation of morphine and codeine to morphinone and codeinone. This enzyme forms the basis of a sensitive detection and assay method for heroin metabolites and a biotransformation process for production of hydromorphone and hydrocodone. To improve these processes we have undertaken a thorough examination of the kinetic mechanism of MDH. Sequence comparisons indicated that MDH belongs within the aldose reductase enzyme family. MDH was shown to be specific for the pro-R hydrogen of NADPH. In steady-state kinetic studies, product inhibition patterns suggested that MDH follows a Theorell-Chance mechanism for codeinone reduction at pH 7, and a non-Theorell-Chance sequential ordered mechanism for codeine oxidation at pH 9.5. Residues corresponding to the catalytically important Tyr-48, Lys-77 and Asp-43 of aldose reductase were modified by site-directed mutagenesis, resulting in substantial loss of activity consistent with a catalytic role for these residues. Loss of activity of MDH in the presence of the reaction product morphinone was found to be due to the formation of a covalent adduct with Cys-80; alteration of Cys-80 to serine resulted in an enzyme with greatly enhanced stability.

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Bioluminescent Assay for Heroin and Its Metabolites

Cited by 18 publications

References 27 publications

Observation of an Arsenic Adduct in an Acetyl Esterase Crystal Structure

Observation of an Arsenic Adduct in an Acetyl Esterase Crystal Structure

Gene Cloning and Nucleotide Sequencing and Properties of a Cocaine Esterase fromRhodococcussp. Strain MB1

Mechanistic studies of morphine dehydrogenase and stabilization against covalent inactivation

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