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Discovery and characterization of a sulfoquinovose mutarotase using kinetic analysis at equilibrium by exchange spectroscopy


Abayakoon, P; Lingford, JP; Jin, Y; Bengt, C; Davies, GJ; Yao, S; Goddard-Borger, ED; Williams, SJ
2018-03-13
Biochemistry Journal
Journal Article
475
7
1371-1383
Bacterial sulfoglycolytic pathways catabolise sulfoquinovose (SQ), or glycosides thereof, to generate a three-carbon metabolite for primary cellular metabolism and a three-carbon sulfonate that is expelled from the cell. Sulfoglycolytic operons encoding an Embden-Meyerhof-Parnas (EMP)-like or Entner-Doudoroff (ED)-like pathway harbour an uncharacterized gene ( yihR in Escherichia coli ; PpSQ1_00415 in Pseudomonas putida ) that is upregulated in the presence of SQ and has been annotated as an aldose-1-epimerase and which may encode an SQ mutarotase. Our sequence analyses and structural modelling confirmed that these proteins possess mutarotase-like active sites with conserved catalytic residues. We over-expressed the homologue from the sulfo-ED operon of Herbaspirillumseropedicaea ( Hs SQM) and used it to demonstrate SQ mutarotase activity for the first time. This was accomplished using NMR exchange spectroscopy (EXSY), a method that allows chemical exchange of magnetization between the two SQ anomers at equilibrium. Hs SQM also catalyzed the mutarotation of various aldohexoses with an equatorial 2-hydroxy group, including D-galactose, D-glucose, D-glucose-6-phosphate, and D-glucuronic acid, but not D-mannose. Hs SQM displayed only 5-fold selectivity in terms of efficiency ( kcat/ KM) for SQ versus the glycolysis intermediate glucose-6-phosphate (Glc-6-P), however its proficiency [ kuncat / ( kcat/ KM)] for SQ was 17,000-fold better than for Glc-6-P, revealing that Hs SQM preferentially stabilises the SQ transition state.
Portland Press
Chemical Biology
10.1042/BCJ20170947
29535276
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