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

Biotechnol Bioeng. 2006 Nov 5;95(4):560-73. doi: 10.1002/bit.20772.

Abstract

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.

MeSH terms

  • Alcohol Oxidoreductases / genetics
  • Alcohol Oxidoreductases / metabolism*
  • Biosynthetic Pathways
  • Gene Expression Regulation, Bacterial
  • Hydro-Lyases / genetics
  • Hydro-Lyases / metabolism*
  • Kinetics
  • 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*
  • Streptococcus pneumoniae / enzymology*

Substances

  • Alcohol Oxidoreductases
  • Shikimate dehydrogenase
  • Phosphotransferases (Alcohol Group Acceptor)
  • shikimate kinase
  • Hydro-Lyases
  • 3-dehydroquinate dehydratase
  • 3-dehydroquinate synthetase
  • Phosphorus-Oxygen Lyases