Subject(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
The avidin-biotin technology has many applications, including molecular detection; immobilization; protein purification; construction of supramolecular assemblies and artificial metalloenzymes. Here we present the recombinant expression of novel biotin-binding proteins from bacteria and the purification and characterization of a secreted burkavidin from the human pathogen Burkholderia pseudomallei. Expression of the native burkavidin in Escherichia coli led to periplasmic secretion and formation of a biotin-binding, thermostable, tetrameric protein containing an intra-monomeric disulphide bond. Burkavidin showed one main species as measured by isoelectric focusing, with lower isoelectric point (pI) than streptavidin. To exemplify the potential use of burkavidin in biotechnology, an artificial metalloenzyme was generated using this novel protein-scaffold and shown to exhibit enantioselectivity in a rhodium-catalysed hydrogenation reaction.
Subject(s)
Bacterial Proteins/metabolism , Biotin/metabolism , Carrier Proteins/metabolism , Hydrogenase/metabolism , Recombinant Proteins/metabolism , Streptavidin/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Burkholderia pseudomallei/chemistry , Carrier Proteins/chemistry , Carrier Proteins/genetics , Catalysis , Cloning, Molecular , Enzyme Stability , Escherichia coli , Humans , Hydrogenase/chemistry , Hydrogenase/genetics , Isoelectric Focusing , Kinetics , Models, Molecular , Molecular Sequence Data , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Rhodium/metabolism , 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.
Subject(s)
Biocatalysis , Directed Molecular Evolution , Proteins/chemistry , Proteins/metabolism , Protein Structure, Secondary , Proteins/genetics , StereoisomerismABSTRACT
The mode of action of precious metal anticancer metallodrugs is generally believed to involve DNA as a target. However, the poor specificity of such drugs often requires high doses and leads to undesirable side-effects. With the aim of improving the specificity of a ruthenium piano-stool complex towards DNA, we employed a presenter protein strategy based on the biotin-avidin technology. Guided by the X-ray structure of the assembly of streptavidin and a biotinylated piano-stool, we explored the formation of metallodrug-mediated ternary complexes with the presenter protein and DNA. The assemblies bound more strongly to telomere G-quadruplexes than to double-stranded DNA; chemo-genetic modifications (varying the complex or mutating the protein) modulated binding to these targets. We suggest that rational targeting of small molecules by presenter proteins could be exploited to bind metallodrugs to preferred macromolecular targets.
