title: 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.
authors.affiliation: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Shekufeh
authors.initials: S
authors.lastname: Zareian
authors.forename: Khosro
authors.initials: K
authors.lastname: Khajeh
authors.forename: Mohammad
authors.initials: M
authors.lastname: Pazhang
authors.forename: Bijan
authors.initials: B
authors.lastname: Ranjbar
mesh_terms: D000494:Allosteric Regulation; D001120:Arginine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019757:Carbon-Oxygen Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013824:Thermus / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D010710:Phosphates; D011994:Recombinant Proteins; D001120:Arginine; D019757:Carbon-Oxygen Lyases; C021578:methylglyoxal synthase