RESUMO
In the first step of this study, type 2 isopentenyl diphosphate isomerase (IDI2) from Pyrococcus furiosus (pf-IDI2), a hyperthermophilic microorganism, was cloned and overexpressed in E. coli. After purification, hyperthermophilic behavior of this protein was approached by means of enzymatic assays and thermal denaturation studies. Compared with the mesophilic Streptococcus pneumoniae IDI2, which unfolds and looses activity above 50 degrees C, pf-IDI2 is still folded and active at 80 degrees C. Molecular modeling was applied, in a parallel step, to understand the molecular basis of thermal stability. Comparison of IDI2 from S. pneumoniae, T. thermophilus, and P. furiosus suggested that additional charged residues present in the hyperthermophilic enzyme might contribute to its higher thermal stability. This could increase the number of salt bridges between monomers of IDI2 in P. furiosus enzyme and, hence, decrease flexibility of loops or N-terminal segment, thereby enhancing its thermal stability.
Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/química , Isomerases de Ligação Dupla Carbono-Carbono/classificação , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Modelos Moleculares , Pyrococcus furiosus/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Isomerases de Ligação Dupla Carbono-Carbono/análise , Isomerases de Ligação Dupla Carbono-Carbono/genética , Isomerases de Ligação Dupla Carbono-Carbono/isolamento & purificação , Clonagem Molecular , Sequência Conservada , Cristalografia por Raios X , Estabilidade Enzimática , Escherichia coli/genética , Hemiterpenos , Temperatura Alta , Ligação de Hidrogênio , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Isopentenyl-diphosphate (IPP):dimethylallyl diphosphate isomerase is a key enzyme in the biosynthesis of isoprenoids. The mechanism of the isomerization reaction involves protonation of the unactivated carbon-carbon double bond in the substrate, but identity of the acidic moiety providing the proton is still not clear. Multiple sequence alignments and geometrical features observed in crystal structures of complexes with IPP isomerase suggest that Tyr-104 could play an important role during catalysis. A series of mutants was constructed by directed mutagenesis and characterized by enzymology. Crystallographic and thermal denaturation data for Y104A and Y104F mutants were obtained. Those data demonstrate the importance of residue Tyr-104 for proper folding of Escherichia coli type I IPP isomerase.
Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/química , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Isomerases de Ligação Dupla Carbono-Carbono/genética , Catálise , Cristalografia , Proteínas de Escherichia coli/genética , Hemiterpenos , Mutagênese Sítio-Dirigida , Desnaturação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Tirosina/metabolismoRESUMO
Driving the equilibrium between selenides and osmium(VIII) reagents with selenoxides and osmium(VI) by a subsequent reaction (rearrangement of allyl selenoxides to allyl alcohols or addition of osmium(VIII) species on C=C double bonds) to one side, allows the transformation of methyl geranyl selenides to linalool and of methyl citronellyl selenoxide to 6,7-dihydroxy citronellyl selenide.