ja508258t_si_001.pdf (1.24 MB)
Structural, Kinetic, and Docking Studies of Artificial Imine Reductases Based on Biotin–Streptavidin Technology: An Induced Lock-and-Key Hypothesis
journal contribution
posted on 2014-11-05, 00:00 authored by Victor
Muñoz Robles, Marc Dürrenberger, Tillmann Heinisch, Agustí Lledós, Tilman Schirmer, Thomas R. Ward, Jean-Didier MaréchalAn
artificial imine reductase results upon incorporation of a biotinylated
Cp*Ir moiety (Cp* = C5Me5–) within homotetrameric streptavidin (Sav) (referred to as Cp*Ir(Biot-p-L)Cl] ⊂ Sav). Mutation of S112 reveals a marked
effect of the Ir/streptavidin ratio on both the saturation kinetics
as well as the enantioselectivity for the production of salsolidine.
For [Cp*Ir(Biot-p-L)Cl] ⊂ S112A Sav, both
the reaction rate and the selectivity (up to 96% ee (R)-salsolidine, kcat 14–4 min–1 vs [Ir], KM 65–370
mM) decrease upon fully saturating all biotin binding sites (the ee
varying between 96% ee and 45% ee R). In contrast,
for [Cp*Ir(Biot-p-L)Cl] ⊂ S112K Sav, both
the rate and the selectivity remain nearly constant upon varying the
Ir/streptavidin ratio [up to 78% ee (S)-salsolidine, kcat 2.6 min–1, KM 95 mM]. X-ray analysis complemented with docking studies
highlight a marked preference of the S112A and S112K Sav mutants for
the SIr and RIr enantiomeric forms of the cofactor, respectively. Combining both
docking and saturation kinetic studies led to the formulation of an
enantioselection mechanism relying on an “induced lock-and-key”
hypothesis: the host protein dictates the configuration of the biotinylated
Ir-cofactor which, in turn, by and large determines the enantioselectivity
of the imine reductase.
History
Usage metrics
Categories
Keywords
enantioselectivityRIr enantiomeric formsDocking Studiesenantioselection mechanismeeselectivitydocking studiesS 112saturation kineticsimine reductase resultskcatS 112K Sav mutantsArtificial Imine ReductasesmMreaction rateKMCpC 5MeS 112Abiotinylatedhomotetrameric streptavidinimine reductasebiotin binding siteshost protein dictates
Licence
Exports
RefWorks
BibTeX
Ref. manager
Endnote
DataCite
NLM
DC