Rationalization of allosteric pathway in Thermus sp. GH5 methylglyoxal synthase
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 sub...
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| Format: | Article |
| Language: | English |
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Korean Society for Biochemistry and Molecular Biology
2012-12-01
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| Series: | BMB Reports |
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| Online Access: | http://www.jbmb.or.kr/jbmb/pdf.php?data=MTMwMTE0MTZAcGRmX3JhaW50cmFjZV9sZWV5c0AlNUI0NS0xMiU1RDEyMTIyNjE2NDZfJTI4NzQ4LTc1MyUyOUJNQl8xMS0xMzgucGRm |
| Summary: | 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. |
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| ISSN: | 1976-6696 1976-670X |