ABSTRACT
High-throughput assays for enzyme catalysis that can be applied to a broad range of chemical reactions are key to advances in directed evolution and proteomics. Recently, we reported such a general assay, chemical complementation, which links enzyme catalysis to reporter gene transcription in vivo using the yeast three-hybrid assay. In this proof-of-principle experiment, it was shown that a wild-type beta-lactamase enzyme could be isolated from a pool of inactive mutants using a lacZ screen. Ideally, however, such an assay should be able to distinguish enzymes based on their catalytic activity. Thus, here, we set out to determine if the catalytic efficiency of an enzyme variant does in fact correlate with its level of transcription activation in the chemical complementation assay. First, the reaction mechanism for the cleavage of the beta-lactam substrate used in the chemical complementation proof-of-principle experiment was determined. Then a series of beta-lactamase variants was designed to span several orders of magnitude in k(cat)/K(m). The activity of each variant was determined both in vitro using purified enzyme and in vivo in the chemical complementation transcription assay. Beta-lactamase variants spanning three-orders of magnitude in k(cat)/K(m) could be distinguished in the assay, and the catalytic efficiency of each variant correlated with its level of transcription activation in vivo. These results establish the suitability of chemical complementation for the directed evolution of enzymes with improvements in catalytic activity and for profiling the relative substrate specificities of a group of enzymes in proteomics applications.
Subject(s)
Transcription, Genetic , beta-Lactamases/chemistry , beta-Lactamases/genetics , Catalysis , Cephalosporins/chemistry , Dexamethasone/chemistry , Directed Molecular Evolution/methods , Enterobacter cloacae/enzymology , Enterobacter cloacae/genetics , Genes, Reporter , Genetic Variation , Hydrolysis , Kinetics , Lac Operon , Methotrexate/chemistry , Mutagenesis, Site-Directed , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Substrate Specificity/genetics , Transcriptional Activation , Two-Hybrid System Techniques , beta-Lactamases/biosynthesis , beta-Lactamases/chemical synthesisSubject(s)
Dexamethasone/pharmacology , Methotrexate/pharmacology , Two-Hybrid System Techniques , Yeasts/genetics , Binding Sites , Gene Expression Regulation/drug effects , Genes, Reporter/genetics , Protein Binding/drug effects , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Yeasts/drug effects , Yeasts/metabolismABSTRACT
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.
