Rationalization of allosteric pathway in Thermus sp. GH5 methylglyoxal synthase

Abstract

A sequence of 10 amino acids at the C-terminus region ofmethylglyoxal synthase from Escherichia coli (EMGS) providesan arginine, which plays a crucial role in forming a salt bridgewith 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 MGSfrom a thermophile species, Thermus sp. GH5 (TMGS) lackingthis arginine was cloned with an additional 30 bp sequence atthe 3´-end and then expressed in form of a fusion TMGS with a10 residual segment at the C-terminus (TMGS+). The resultingrecombinant enzyme showed a significant increase in cooperativitytowards phosphate, reflected by a change in the Hillcoefficient (nH) from 1.5 to 1.99. Experiments including sitedirected mutagenesis for Asp-10 in TMGS and TMGS+, twodimentional structural survey, fluorescence and irreversiblethermoinactivation were carried out to confirm this pathway.

Keywords

• Allosteric pathway
• Cooperativity
• Hill coefficient
• Methylglyoxal synthase
• Structural compactness