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

Bacterial sulfoglycolytic pathways catabolize 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-like or Entner-Doudoroff (ED)-like pathway harbor an uncharacterized gene ( yihR in Escherichia coli ; PpSQ1_00415 in Pseudomonas putida ) that is up-regulated in the presence of SQ, has been annotated as an aldose-1-epimerase and which may encode an SQ mutarotase. Our sequence analyses and structural modeling confirmed that these proteins possess mutarotase-like active sites with conserved catalytic residues. We overexpressed the homolog from the sulfo-ED operon of Herbaspirillum seropedicaea ( Hs SQM) and used it to demonstrate SQ mutarotase activity for the first time. This was accomplished using nuclear magnetic resonance exchange spectroscopy, a method that allows the 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 (Glc-6-P), and d-glucuronic acid, but not d-mannose. Hs SQM displayed only 5-fold selectivity in terms of efficiency ( k cat / K M ) for SQ versus the glycolysis intermediate Glc-6-P; however, its proficiency [ k uncat /( k cat / K M )] for SQ was 17 000-fold better than for Glc-6-P, revealing that Hs SQM preferentially stabilizes the SQ transition state.