Rationalization of allosteric pathway in Thermus sp. GH5 methylglyoxal synthase

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.

Fig. 1.. Michaelis-Menten curve and Hill plot of four variants in different concentrations of DHAP as substrate. (A) 0 mM phosphate, (B) 1.5 mM phosphate (C) Hill plot of four variants. Hill plot was drawn according to the equation mentioned in the Materials and Methods section, in the presence of 1.5 mM of phosphate. (♦) TMGS, (▲) TMGS D10N, (■) TMGS⁺, (●) TMGS⁺D10N.

Fig. 2.. Far-UV CD spectra of TMGS (- - - -) and TMGS⁺ (−). Diagram reveals a more helical structure for TMGS⁺.

Fig. 3.. Fluorescence emission spectra of TMGS (- - - -) and TMGS⁺ (−) at 280 nm excitation wavelengths, the inset is the spectra for the excitation at 293 nm. Graphs represent more compact structure for TMGS⁺.