title: Biochemical characterization and crystallographic structure of an Escherichia coli protein from the phosphotriesterase gene family.
abstract: Phosphotriesterase homology protein (PHP) is a member of a recently discovered family of proteins related to phosphotriesterase, a hydrolytic, bacterial enzyme with an unusual substrate specificity for synthetic organophosphate triesters and phosphorofluoridates, which are common constituents of chemical warfare agents and agricultural pesticides. No natural substrate has been identified for phosphotriesterase, and it has been suggested that the enzyme may have evolved the ability to hydrolyze synthetic compounds in bacteria under selective pressure to meet nutritional needs. PHP, which has 28% sequence identity with phosphotriesterase, may belong to the family of proteins from which phosphotriesterase evolved. Here we report the cloning, expression, initial characterization, and high-resolution X-ray crystallographic structure of PHP. Biochemical analysis shows that PHP is monomeric and binds two zinc ions per monomer. Unlike phosphotriesterase, PHP does not catalyze the hydrolysis of nonspecific phosphotriesters. The structure, similar to that of phosphotriesterase, consists of a long, elliptical alpha/beta barrel and has a binuclear zinc center in a cleft at the carboxy end of the barrel at the location of the presumptive active site.
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143-0448, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Buchbinder
authors.forename: R C
authors.initials: RC
authors.lastname: Stephenson
authors.forename: M J
authors.initials: MJ
authors.lastname: Dresser
authors.forename: J W
authors.initials: JW
authors.lastname: Pitera
authors.forename: T S
authors.initials: TS
authors.lastname: Scanlan
authors.forename: R J
authors.initials: RJ
authors.lastname: Fletterick
mesh_terms: D000595:Amino Acid Sequence; D043303:Aryldialkylphosphatase; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D004950:Esterases; D019143:Evolution, Molecular; D005798:Genes, Bacterial; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008667:Metalloproteins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014305:Triose-Phosphate Isomerase / Q000737:chemistry; D015032:Zinc / Q000032:analysis
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008667:Metalloproteins; D011994:Recombinant Proteins; D006867:Hydrolases; D004950:Esterases; C111817:PHP protein, E coli; D043303:Aryldialkylphosphatase; D014305:Triose-Phosphate Isomerase; D015032:Zinc