ABSTRACT
A novel solid-phase screening assay was developed for colonies expressing both (R)- and (S)-selective ω-aminotransferases. This high-throughput assay can be used to screen rapidly large variant libraries with enhanced substrate selectivity and enantioselectivities.
Subject(s)
Bacteria/classification , Bacteria/enzymology , Bacterial Typing Techniques/methods , Biological Assay/methods , High-Throughput Screening Assays/methods , Bacteria/isolation & purification , Stereoisomerism , TransaminasesABSTRACT
Atropisomeric biaryls carrying ortho-hydroxymethyl and formyl groups were made enantioselectively by desymmetrisation of dialdehyde or diol substrates. The oxidation of the symmetrical diol substrates was achieved using a variant of galactose oxidase (GOase), and the reduction of the dialdehydes using a panel of ketoreductases. Either M or P enantiomers of the products could be formed, with absolute configurations assigned by time-dependent DFT calculations of circular dichroism spectra. The differing selectivities observed with different biaryl structures offer an insight into the detailed structure of the active site of the GOase enzyme.
Subject(s)
Galactose Oxidase/metabolism , Oxidoreductases/metabolism , Aldehydes/chemistry , Aldehydes/metabolism , Biocatalysis , Catalytic Domain , Circular Dichroism , Galactose Oxidase/chemistry , Galactose Oxidase/genetics , Models, Molecular , Mutation , Oxidation-Reduction , Oxidoreductases/chemistry , StereoisomerismABSTRACT
The development of cost-effective and sustainable catalytic methods for the production of enantiomerically pure chiral amines is a key challenge facing the pharmaceutical and fine chemical industries. This challenge is highlighted by the estimate that 40-45% of drug candidates contain a chiral amine, fueling a demand for broadly applicable synthetic methods that deliver target structures in high yield and enantiomeric excess. Herein we describe the development and application of a "toolbox" of monoamine oxidase variants from Aspergillus niger (MAO-N) which display remarkable substrate scope and tolerance for sterically demanding motifs, including a new variant, which exhibits high activity and enantioselectivity toward substrates containing the aminodiphenylmethane (benzhydrylamine) template. By combining rational structure-guided engineering with high-throughput screening, it has been possible to expand the substrate scope of MAO-N to accommodate amine substrates containing bulky aryl substituents. These engineered MAO-N biocatalysts have been applied in deracemization reactions for the efficient asymmetric synthesis of the generic active pharmaceutical ingredients Solifenacin and Levocetirizine as well as the natural products (R)-coniine, (R)-eleagnine, and (R)-leptaflorine. We also report a novel MAO-N mediated asymmetric oxidative Pictet-Spengler approach to the synthesis of (R)-harmicine.
Subject(s)
Alkaloids/metabolism , Aspergillus niger/enzymology , Monoamine Oxidase/genetics , Protein Engineering/methods , Alkaloids/chemistry , Aspergillus niger/chemistry , Aspergillus niger/genetics , Aspergillus niger/metabolism , Benzhydryl Compounds/chemistry , Benzhydryl Compounds/metabolism , Biological Products/chemistry , Biological Products/metabolism , Indole Alkaloids/chemistry , Indole Alkaloids/metabolism , Models, Molecular , Monoamine Oxidase/metabolism , Oxidation-Reduction , StereoisomerismABSTRACT
A directed evolution approach has been used for the generation of variants of galactose oxidase (GOase) that can selectively oxidize glycans on glycoproteins. The aldehyde function introduced on the glycans D-mannose (Man) and D-N-acetyl glucosamine (GlcNAc) by the enzyme variants could then be used to label the glycoproteins and also whole cells that display mannosides on their surface.
Subject(s)
Chromogenic Compounds/chemistry , Evolution, Molecular , Galactose Oxidase/genetics , Glycoproteins/genetics , Galactose Oxidase/chemistry , Genetic Variation , Glycoproteins/chemistry , Models, Molecular , Molecular Structure , Protein Engineering , Substrate SpecificityABSTRACT
The crystal structure of Escherichia coli bacterioferritin has been solved to 1.9 A, and shows the symmetrical binding of a haem molecule on the local twofold axis between subunits and a pair of metal atoms bound to each subunit at the ferroxidase centre. These metals have been identified as zinc by the analysis of the structure and X-ray data and confirmed by microfocused proton-induced X-ray emission experiments. For the first time the haem has been shown to be linked to both the internal and the external environments via a cluster of waters positioned above the haem molecule.
