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http://dx.doi.org/10.5483/BMBRep.2012.45.12.11-138

Rationalization of allosteric pathway in Thermus sp. GH5 methylglyoxal synthase  

Zareian, Shekufeh (Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University)
Khajeh, Khosro (Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University)
Pazhang, Mohammad (Department of Cellular and Molecular Biology, Faculty of Science, Azarbaijan University of Tarbiat Moallem)
Ranjbar, Bijan (Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University)
Publication Information
BMB Reports / v.45, no.12, 2012 , pp. 748-753 More about this Journal
Abstract
A sequence of 10 amino acids at the C-terminus region of methylglyoxal synthase from Escherichia coli (EMGS) provides an arginine, which plays a crucial role in forming a salt bridge with a proximal aspartate residue in the neighboring subunit, consequently transferring the allosteric signal between subunits. In order to verify the role of arginine, the gene encoding MGS from a thermophile species, Thermus sp. GH5 (TMGS) lacking this arginine was cloned with an additional 30 bp sequence at the 3'-end and then expressed in form of a fusion TMGS with a 10 residual segment at the C-terminus ($TMGS^+$). The resulting recombinant enzyme showed a significant increase in cooperativity towards phosphate, reflected by a change in the Hill coefficient (nH) from 1.5 to 1.99. Experiments including site directed mutagenesis for Asp-10 in TMGS and $TMGS^+$, two dimentional structural survey, fluorescence and irreversible thermoinactivation were carried out to confirm this pathway.
Keywords
Allosteric pathway; Cooperativity; Hill coefficient; Methylglyoxal synthase; Structural compactness;
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