RESUMO
Production of fine chemicals from heterologous pathways in microbial hosts is frequently hindered by insufficient knowledge of the native metabolic pathway and its cognate enzymes; often the pathway is unresolved, and the enzymes lack detailed characterization. An alternative paradigm to using native pathways is de novo pathway design using well-characterized, substrate-promiscuous enzymes. We demonstrate this concept using P450(BM3) from Bacillus megaterium. Using a computer model, we illustrate how key P450(BM3) active site mutations enable binding of the non-native substrate amorphadiene. Incorporating these mutations into P450(BM3) enabled the selective oxidation of amorphadiene artemisinic-11S,12-epoxide, at titers of 250 mg L(-1) in E. coli. We also demonstrate high-yielding, selective transformations to dihydroartemisinic acid, the immediate precursor to the high-value antimalarial drug artemisinin.
Assuntos
Artemisininas/metabolismo , Bacillus megaterium/enzimologia , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Engenharia de Proteínas , Algoritmos , Artemisininas/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Simulação por Computador , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Modelos Moleculares , Conformação Molecular , Mutação , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/genética , Oxirredução , Sesquiterpenos Policíclicos , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Estereoisomerismo , Fatores de TempoRESUMO
A small library of 19 compounds was designed based on unique structural features of PTP1b. Utilizing electrospray ionization mass spectrometry (ESI-MS) to provide binding information about complexes of enzyme and small molecule ligands, two classes of lead compounds were discovered.
Assuntos
Inibidores Enzimáticos/química , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteínas Tirosina Fosfatases/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
A novel class of nonpeptide inhibitors of stromelysin (MMP-3) has been discovered with the use of mass spectrometry. The method relies on the development of structure-activity relationships by mass spectrometry (SAR by MS) and utilizes information derived from the binding of known inhibitors to identify novel inhibitors of a target protein with a minimum of synthetic effort. Noncovalent complexes of known inhibitors with a target protein are analyzed; these inhibitors are deconstructed into sets of fragments which compete for common or overlapping binding sites on the target protein. The binding of each fragment set can be studied independently. With the use of competition studies, novel members of each fragment set are identified from compound libraries that bind to the same site on the target protein. A novel inhibitor of the target protein was then constructed by chemically linking a combination of members of each fragment set in a manner guided by the proximity and orientation of the fragments derived from the known inhibitors. In the case of stromelysin, a novel inhibitor composed of favorably linked fragments was observed to form a 1:1 complex with stromelysin. Compounds that were not linked appropriately formed higher order complexes with stoichiometries of 2:1 or greater. These linked molecules were subsequently assessed for their ability to block stromelysin function in a chromogenic substrate assay.