ABSTRACT
We report our efforts to enable transition-metal catalysis in the presence of cellular debris, notably Escherichia coli cell free extracts and cell lysates. This challenging goal is hampered by the presence of thiols, mainly present in the form of glutathione (GSH), which poison precious metal catalysts. To overcome this, we evaluated a selection of oxidizing agents and electrophiles toward their potential to neutralize the detrimental effect of GSH on a Ir-based transfer hydrogenation catalyst. While the bare catalyst was severely inhibited by cellular debris, embedding the organometallic moiety within a host protein led to promising results in the presence of some neutralizing agents. In view of its complementary to natural enzymes, the asymmetric imine reductase based on the incorporation of a biotinylated iridium pianostool complex within streptavidin (Sav) isoforms was selected as a model reaction. Compared to purified protein samples, we show that pretreatment of cell free extracts and cell lysates containing Sav mutants with diamide affords up to >100 TON's and only a modest erosion of enantioselectivity.
Subject(s)
Glutathione/chemistry , Iridium/chemistry , Organometallic Compounds/chemistry , Biocatalysis , Catalysis , Escherichia coli/cytology , Escherichia coli/metabolism , Glutathione/metabolism , Iridium/metabolism , Models, Molecular , Molecular Structure , Organometallic Compounds/metabolism , Oxidoreductases/metabolismABSTRACT
Alternating: a cofactor dyad consisting of a heme group (red in picture) and a bis(biotin) unit (blue) was synthesized and shown to specifically bind to both apomyoglobin and streptavidin. In the presence of the dyad, the 1:1 association of a disulfide-bridged myoglobin dimer (green) with streptavidin (gray) afforded a submicrometer-sized fibrous alternating copolymer.
Subject(s)
Hemeproteins/chemistry , Streptavidin/chemistry , Binding Sites , Biotin/chemistry , Biotin/metabolism , Hemeproteins/metabolism , Myoglobin/chemistry , Myoglobin/metabolism , Streptavidin/metabolismSubject(s)
Alkenes/chemistry , Osmium Tetroxide/chemistry , Streptavidin/chemistry , Amino Acid Substitution , Binding Sites , Biocatalysis , Crystallography, X-Ray , Dioxygenases/genetics , Dioxygenases/metabolism , Hydroxylation , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Protein Structure, Tertiary , StereoisomerismABSTRACT
Artificial metalloenzymes result from the introduction of a catalytically competent non-native metal cofactor within a protein environment. In the present contribution, we summarize the recent achievements in the design and the optimization of such protein-based hybrid catalysts, with an emphasis on enantioselective transformations. The second part outlines the milestones required to achieve en masse production, screening and directed evolution of artificial metalloenzymes. In the spirit of Darwinian evolution, this will allow the full potential of such protein-based hybrid catalysts to be fully unraveled, thus complementing both homogeneous and enzymatic catalysis.
