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

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.

Identification of Polyketide Inhibitors Targeting 3-Dehydroquinate Dehydratase in the Shikimate Pathway of Enterococcus faecalis

“…This is comparable to DHQases from other type I DHQases such as Escherichia coli [28], Salmonella typhi [7], and Streptococcus pneumoniae [29] characterized under similar conditions (Table 2). …”

Insights into the Mechanism of Type I Dehydroquinate Dehydratases from Structures of Reaction Intermediates

“…Reaction was initiated by the addition of protein to a mixture of buffer and 3-dehydroquinic acid (Sigma-Aldrich). Formation of the conjugated enone carboxylate in dehydroshikimate was followed by measuring the increase in absorbance at ϭ 234 nm relative to substrate (⑀ ϭ 12 mM Ϫ1 cm Ϫ1 ) (4,19). To determine the kinetic parameters, triplicate measurements of the reaction rate were determined at varying concentrations of 3-dehydroquinic acid.…”

“…Chorismate serves as a precursor of many biologically important aromatic compounds including the ubiquinones, folates, and aromatic amino acids (1). The essential nature of these proteins in a number of species, in combination with an absence of human homologs, makes the shikimate pathway an attractive target for the development of non-toxic antimicrobials, anti-fungals, and herbicides (2)(3)(4). Given the complexities inherent to inhibitor design, a comprehensive understanding of the structural and mechanistic framework underlying the function of the shikimate pathway enzymes should aid in the development of novel shikimate-targeting inhibitors.…”

Genome-Wide Identification of Genes Essential for the Survival of Streptococcus pneumoniae in Human Saliva

“…In a murine model of pneumococcal colonization, deletion of ami genes resulted in pneumococci that were less able to colonize the nasopharynx [28]. While the oligopeptide transport systems are involved in the uptake of nutrients from the external environment, the aro genes are involved in the biosynthesis of aromatic amino acids [29]. Since the concentration of amino acids in saliva is low [15], their biosynthesis is likely to become more important, which explains the observed decrease in survival of the aro mutants in our screen.…”