Correlation between catalytic efficiency and the transcription read-out in chemical complementation: a general assay for enzyme catalysis.
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.
Sengupta
Debleena
D
Department of Chemistry, Columbia University, New York, New York 10027, USA.
Lin
Hening
H
Goldberg
Shalom D
SD
Mahal
Jacqueline J
JJ
Cornish
Virginia W
VW
D002384:Catalysis; D002511:Cephalosporins / Q000737:chemistry; D003907:Dexamethasone / Q000737:chemistry; D019020:Directed Molecular Evolution / Q000379:methods; D016972:Enterobacter cloacae / Q000201:enzymology / Q000235:genetics; D017930:Genes, Reporter; D014644:Genetic Variation; D006868:Hydrolysis; D007700:Kinetics; D007763:Lac Operon; D008727:Methotrexate / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques; D001618:beta-Lactamases / Q000096:biosynthesis / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics*
D002511:Cephalosporins; D011994:Recombinant Proteins; D003907:Dexamethasone; D001618:beta-Lactamases; D008727:Methotrexate