title: Chemical complementation: a reaction-independent genetic assay for enzyme catalysis.
abstract: A high-throughput assay for enzyme activity has been developed that is reaction independent. In this assay, a small-molecule yeast three-hybrid system is used to link enzyme catalysis to transcription of a reporter gene in vivo. Here we demonstrate the feasibility of this approach by using a well-studied enzyme-catalyzed reaction, cephalosporin hydrolysis by the Enterobacter cloacae P99 cephalosporinase (beta-lactam hydrolase, EC ). We show that the three-hybrid system can be used to read out cephalosporinase activity in vivo as a change in the level of transcription of a lacZ reporter gene and that the wild-type cephalosporinase can be isolated from a pool of inactive mutants by using a lacZ screen. The assay has been designed so that it can be applied to different chemical reactions without changing the components of the three-hybrid system. A reaction-independent high-throughput assay for protein function should be a powerful tool for protein engineering and enzymology, drug discovery, and proteomics.
authors.affiliation: Departments of Chemistry and Pharmacology, Columbia University, New York, NY 10027, USA.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Baker
authors.forename: Colleen
authors.initials: C
authors.lastname: Bleczinski
authors.forename: Hening
authors.initials: H
authors.lastname: Lin
authors.forename: Gilda
authors.initials: G
authors.lastname: Salazar-Jimenez
authors.forename: Debleena
authors.initials: D
authors.lastname: Sengupta
authors.forename: Sonja
authors.initials: S
authors.lastname: Krane
authors.forename: Virginia W
authors.initials: VW
authors.lastname: Cornish
mesh_terms: D002384:Catalysis; D002510:Cephalosporinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002511:Cephalosporins / Q000378:metabolism; D019281:Dimerization; D016972:Enterobacter cloacae / Q000201:enzymology*; D017930:Genes, Reporter; D006868:Hydrolysis; D007763:Lac Operon; D015202:Protein Engineering; D014158:Transcription, Genetic
chemical_list: D002511:Cephalosporins; D002510:Cephalosporinase