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 (␤-lactam hydrolase, EC 3.5.2.6). 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 threehybrid system. A reaction-independent high-throughput assay for protein function should be a powerful tool for protein engineering and enzymology, drug discovery, and proteomics. E nzymes are able to catalyze a broad range of chemical transformations not only with impressive rate enhancements but also with both regio-and stereoselectivity and so are attractive candidates as practical alternatives to traditional smallmolecule catalysts. With applications as diverse as chemical synthesis, reagents for commercial products and biomedical research, and even therapeutics, there is a great demand for enzymes with both improved activity and novel catalytic function (1, 2). In theory, the properties of an enzyme can be altered by rational design; however, rational design is greatly hindered in practice by the complexity of protein function. With advances in molecular biology the possibility has arisen that an enzyme with the desired catalytic property can instead be isolated from a large pool of protein variants. Recently directed evolution has been used successfully to modify the substrate (3) or cofactor specificity (4) of an existing enzyme. These experiments, however, are limited to reactions that are inherently screenable or selectable, reactions where the substrate is a peptide (5, 6) or the product is fluorescent (4) or an essential metabolite (3,7,8). What are needed now to realize the power of directed evolution experiments are screening and selection strategies that are general, strategies that do not limit the chemistry and that can readily be adapted to a new target reaction.Early success with assays based on binding to transition-state analogs and suicide substrates convinced researchers that it should be possible to engineer proteins to catalyze a broad range of reactions (9), but it was difficult to translate binding events into read-outs for enhanced catalytic activity. Recently, attention has turned to direct selections for catalytic activity. While strategies ranging from in vitro fluorescence assays to physically linking the enzyme to its substrate have all recently been reported (10-15), a general solution to this problem is yet to emerge.In vivo complementation, in which a...

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