The kinetic model of the shikimate pathway as a tool to optimize enzyme assays for high-throughput screening

• Published in: Biotechnology and bioengineering Vol. 95; no. 4; pp. 560 - 571
• Main Authors: Noble, Michael, Sinha, Yugesh, Kolupaev, Aleksey, Demin, Oleg, Earnshaw, David, Tobin, Frank, West, Joshua, Martin, John D., Qiu, Chunyan, Liu, Wu-Schyong, DeWolf Jr, Walter E., Tew, David, Goryanin, Igor I.
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 05.11.2006; Wiley Subscription Services, Inc
• Subjects: Alcohol Oxidoreductases - genetics; Alcohol Oxidoreductases - metabolism; Biosynthetic Pathways; Enzyme kinetics; Gene Expression Regulation, Bacterial; Hydro-Lyases - genetics; Hydro-Lyases - metabolism; Kinetics; Medical screening; Models, Biological; Molecular Biology - methods; Oligonucleotide Array Sequence Analysis; Phosphorus-Oxygen Lyases - genetics; Phosphorus-Oxygen Lyases - metabolism; Phosphotransferases (Alcohol Group Acceptor) - genetics; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Protein synthesis; Streptococcus infections; Streptococcus pneumoniae; Streptococcus pneumoniae - enzymology
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.20772

Four‐enzyme section of the shikimate pathway (Aro B, D, E, and K) of Streptococcus pneumoniae has been studied. Kinetic properties of the individual enzymes and three‐ and four‐enzyme linked reactions have been characterized in vitro. On the basis of the data measured in spectrophotometric and LC‐MS experiments, kinetic mechanisms of the enzymes have been suggested and all kinetic parameters have been identified. Kinetic models for these three‐ and four‐enzyme sections of the shikimate pathway have been constructed and validated. The model of the four‐enzyme section of shikimate pathway has been employed to design an inhibition‐sensitive reconstituted pathway for a high‐throughput screening effort on the shikimate pathway. It was demonstrated that using the model it was possible to optimize this reconstituted pathway in such a way to provide equal sensitivity of the enzymes to inhibition. © 2006 Wiley Periodicals, Inc.