RESUMO
To enhance the activity of transketolase towards nonphosphorylated substrates and enlarge the scope of its substrates, notably to long polyol aldehyde acceptors (D-ribose or D-glucose), a rational design-supported evolution strategy was applied. By using docking experiments, an in silico library, and iterative mutagenesis, libraries of single- and double-point mutants were designed and generated. A double-screening approach was implemented, coupling a preselection activity assay (HPLC method) and a selective assay (GC method) to find the best enzymes. Several mutants (R526N, R526Q, R526Q/S525T, R526K/S525T) showed improved activities towards nonphosphorylated substrates as the coupled products of lithium hydroxypyruvate (HPA) with glycolaldehyde (GO), D-ribose or D-glucose. These mutated enzymes were further characterised. They were shown to be up to four times more active than the wild-type (mutant R526Q/S525T) for nonphosphorylated substrates LiHPA/GO (V(m) /K(m) for LiHPA = 92.4 instead of 28.8×10(-3) min(-1) for the wild-type) and 2.6 times more active for substrates LiHPA/rib.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Engenharia de Proteínas , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transcetolase/genética , Acetaldeído/análogos & derivados , Acetaldeído/metabolismo , Biologia Computacional , Evolução Molecular Direcionada , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Glucose/metabolismo , Modelos Moleculares , Mutação , Piruvatos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribose/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Transcetolase/metabolismoRESUMO
Cascade synthetic pathways, which allow multi-step conversions to take place in one reaction vessel, are crucial for the development of biomimetic, highly efficient new methods of chemical synthesis. Theoretically, the complexity introduced by combining processes could lead to an improvement of the overall process; however, it is the current general belief that it is more efficient to run processes separately. Inspired by natural cascade procedures we successfully combined a lipase catalyzed amidation with palladium catalyzed coupling reactions, simultaneously carried out on the same molecule. Unexpectedly, the bio- and chemo-catalyzed processes show synergistic behaviour, highlighting the complexity of multi-catalyst systems.
Assuntos
Biocatálise , Amidas/química , Aminação , Benzilaminas/química , Benzilaminas/metabolismo , Biomimética , Proteínas Fúngicas , Lipase/química , Lipase/metabolismo , Paládio/químicaRESUMO
[reaction: see text] A new class of cyclohexane-based P,N-ligands is readily obtained through aziridine ring opening with suitable phosphorus nucleophiles under acidic conditions. trans-1-Amino-2-diphenylphosphinocyclohexane is resolved with tartaric acid to give the final product in >99% ee. The new ligands show high stability toward oxidation at the phosphorus atom.
RESUMO
While investigating the antioxidant properties of aminoethylcysteine ketimine decarboxylated dimer (1) (a natural substance occurring in biological fluids such as human urine and plasma and in bovine cerebellum), a previously unreported oxidation product was obtained. This compound was identified and characterized through comparison with an authentic sample prepared via Pd-catalyzed dehydrogenation of 1. This molecule is an example of an alternative oxidation pathway involving 1.