Crystal structure of a bacterial cocaine esterase

Here we report the first structure of a cocaine-degrading enzyme. The bacterial esterase, cocE, hydrolyzes pharmacologically active (−)-cocaine to a nonpsychoactive metabolite with a rate faster than any other reported cocaine esterase (kcat = 7.8 s−1 and KM = 640 nM). Because of the high catalytic proficiency of cocE, it is an attractive candidate for novel protein-based therapies for cocaine overdose. The crystal structure of cocE, solved by multiple anomalous dispersion (MAD) methods, reveals that cocE is a serine esterase composed of three domains: (i) a canonical α/β hydrolase fold (ii) an α-helical domain that caps the active site and (iii) a jelly-roll-like β-domain that interacts extensively with the other two domains. The active site was identified within the interface of all three domains by analysis of the crystal structures of transition state analog adduct and product complexes, which were refined at 1.58 Å and 1.63 Å resolution, respectively. These structural studies suggest that substrate recognition arises partly from interactions between the benzoyl moiety of cocaine and a highly evolved specificity pocket.

[1]  J Zhang,et al.  Purification and Cloning of a Broad Substrate Specificity Human Liver Carboxylesterase That Catalyzes the Hydrolysis of Cocaine and Heroin* , 1997, The Journal of Biological Chemistry.

[2]  J. L. Smith,et al.  Structure of myohemerythrin in the azidomet state at 1.7/1.3 A resolution. , 1987, Journal of molecular biology.

[3]  M. Kuhar,et al.  Pharmacotherapies for treatment of cocaine abuse: preclinical aspects. , 1999, Journal of medicinal chemistry.

[4]  G. Sheldrick,et al.  SHELXL: high-resolution refinement. , 1997, Methods in enzymology.

[5]  K D Cowtan,et al.  Phase combination and cross validation in iterated density-modification calculations. , 1996, Acta crystallographica. Section D, Biological crystallography.

[6]  D. Landry,et al.  A catalytic antibody against cocaine prevents cocaine's reinforcing and toxic effects in rats. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[7]  T. Terwilliger,et al.  Bayesian correlated MAD phasing. , 1997, Acta crystallographica. Section D, Biological crystallography.

[8]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[9]  R. Read.  Improved Fourier Coefficients for Maps Using Phases from Partial Structures with Errors , 1986 .

[10]  W. Minor,et al.  Structure of a microbial homologue of mammalian platelet-activating factor acetylhydrolases: Streptomyces exfoliatus lipase at 1.9 A resolution. , 1998, Structure.

[11]  Z. Derewenda,et al.  Molecular mechanism of enantiorecognition by esterases , 1995 .

[12]  N. Bruce,et al.  Gene Cloning and Nucleotide Sequencing and Properties of a Cocaine Esterase from Rhodococcus sp. Strain MB1 , 2000, Applied and Environmental Microbiology.

[13]  Joel L. Sussman,et al.  The α/β hydrolase fold , 1992 .

[14]  E A Merritt,et al.  Raster3D Version 2.0. A program for photorealistic molecular graphics. , 1994, Acta crystallographica. Section D, Biological crystallography.

[15]  Thomas C. Terwilliger,et al.  Electronic Reprint Biological Crystallography Maximum-likelihood Density Modification , 2022 .

[16]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[17]  W. Kalow,et al.  Metabolism of cocaine in man , 1978, Clinical pharmacology and therapeutics.

[18]  W. Bosron,et al.  Purification and characterization of a human liver cocaine carboxylesterase that catalyzes the production of benzoylecgonine and the formation of cocaethylene from alcohol and cocaine. , 1994, Biochemical pharmacology.

[19]  H. van Tilbeurgh,et al.  The 2.46 A resolution structure of the pancreatic lipase-colipase complex inhibited by a C11 alkyl phosphonate. , 1994, Biochemistry.

[20]  R M Esnouf,et al.  Further additions to MolScript version 1.4, including reading and contouring of electron-density maps. , 1999, Acta crystallographica. Section D, Biological crystallography.

[21]  J. Cashman,et al.  An improved cocaine hydrolase: the A328Y mutant of human butyrylcholinesterase is 4-fold more efficient. , 1999, Molecular pharmacology.

[22]  M. Nardini,et al.  α/β Hydrolase fold enzymes : the family keeps growing , 1999 .

[23]  R. Sherwood,et al.  Antibody-enzyme conjugates for cancer therapy. , 1996, Journal of the National Cancer Institute.

[24]  C. Lowe,et al.  Identification of a cocaine esterase in a strain of Pseudomonas maltophilia , 1992, Journal of bacteriology.

[25]  M. L. Connolly.  Analytical molecular surface calculation , 1983 .

[26]  László Polgár,et al.  Prolyl Oligopeptidase An Unusual β-Propeller Domain Regulates Proteolysis , 1998, Cell.

[27]  V S Lamzin,et al.  Automated refinement of protein models. , 1993, Acta crystallographica. Section D, Biological crystallography.

[28]  L. Parsons,et al.  Suppression of psychoactive effects of cocaine by active immunization , 1995, Nature.

[29]  D. Landry,et al.  Antibody-catalyzed degradation of cocaine. , 1993, Science.

[30]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[31]  A. Goldman,et al.  Of barn owls and bankers: a lush variety of α/β hydrolases , 1999 .

[32]  C. Sander,et al.  Protein structure comparison by alignment of distance matrices. , 1993, Journal of molecular biology.