Selective probing of a NADPH site controlled light-induced enzymatic catalysis

Abstract : Achieving molecular recognition of NADPH binding sites is a compelling strategy to control many redox biological processes. The NADPH sites recognize the ubiquitous NADPH cofactor via highly conserved binding interactions, despite differences in the regulation of the hydride transfer in redox active proteins. We recently developed a photoactive NADPH substitute, called nanotrigger NT synchronizing the initiation of enzymatic catalysis of the endothelial NO-synthase (eNOS) with a laser pulse. Spatial and temporal control of enzymatic activity by such a designed light-driven activator would benefit from achieving molecular selectivity, i.e. activation of a single NADPH-mediated enzyme. In this work, we probe the ability of NT to discriminate between two NADPH sites with light. The selected NADPH sites belong to dihydrofolate reductase dihydrofolate reductase enzyme (DHFR) and endothelial NO-synthase (eNOS). Ultrafast kinetics showed that NT could not activate DHFR catalysis with a laser pulse in contrast with the observed trigger of eNOS catalysis leading to NO formation. Homology modelling, molecular dynamics simulations showed that NT discriminated between the two NADPH sites by different donor to acceptor distances and by local steric effects hindering light activation of DHFR catalysis. The data suggested that the narrow NADPH site required a tight fit of the nanotrigger at a suitable distance/angle to the electron acceptor for a specific activation of the catalysis. The ability of the nanotrigger to activate eNOS combined with a low reactivity in unfavourable NADPH sites makes NT a highly promising tool for targeting eNOS in endothelial cells with a laser pulse. Cop 2009 John Wiley and Sons, Ltd.
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Jean-Christophe Lambry, Edward Beaumont, Bogdan Tarus, Mireille Blanchard-Desce, Anny Slama-Schwok. Selective probing of a NADPH site controlled light-induced enzymatic catalysis. Journal of Molecular Recognition, Wiley, 2010, 23 (4), pp.379. ⟨10.1002/jmr.1009⟩. ⟨hal-00807882⟩

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