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Overexpression, physicochemical characterization, and modeling of a hyperthermophilic pyrococcus furiosus type 2 IPP isomerase
Author(s) -
Dutoit Raphaël,
de Ruyck Jérôme,
Durisotti Virginie,
Legrain Christianne,
Jacobs Eric,
Wouters Johan
Publication year - 2008
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.21863
Subject(s) - pyrococcus furiosus , thermophile , thermostability , chemistry , thermal stability , isomerase , thermus thermophilus , enzyme , denaturation (fissile materials) , mesophile , biochemistry , bacteria , escherichia coli , biology , organic chemistry , gene , archaea , nuclear chemistry , genetics
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°C, pf‐IDI2 is still folded and active at 80°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. Proteins 2008. © 2007 Wiley‐Liss, Inc.