title: Overexpression, physicochemical characterization, and modeling of a hyperthermophilic pyrococcus furiosus type 2 IPP isomerase.
abstract: 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.
authors: Institut de Recherches Microbiologiques J.M. Wiame, 1 av E. Gryson, 1070 Bruxelles, Belgium.
authors: Raphaël
authors: R
authors: Dutoit
authors: Jérôme
authors: J
authors: de Ruyck
authors: Virginie
authors: V
authors: Durisotti
authors: Christianne
authors: C
authors: Legrain
authors: Eric
authors: E
authors: Jacobs
authors: Johan
authors: J
authors: Wouters
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D019748:Carbon-Carbon Double Bond Isomerases / Q000032:analysis / Q000737:chemistry* / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D045782:Hemiterpenes; D006358:Hot Temperature; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D020027:Pyrococcus furiosus / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D045782:Hemiterpenes; D011994:Recombinant Proteins; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase