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

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Author(s)

  • Palika Abayakoon
  • James P. Lingford
  • Yi Jin
  • Christopher Bengt
  • Gideon J. Davies
  • Shenggen Yao
  • Ethan D. Goddard-Borger
  • Spencer J. Williams

Department/unit(s)

Publication details

JournalBiochemical journal
DateAccepted/In press - 12 Mar 2018
DateE-pub ahead of print - 13 Mar 2018
DatePublished (current) - 16 Apr 2018
Issue number7
Volume475
Number of pages13
Pages (from-to)1371-1383
Early online date13/03/18
Original languageEnglish

Abstract

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 (HsSQM) 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. HsSQM 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. HsSQM displayed only 5-fold selectivity in terms of efficiency (kcat/KM) for SQ versus the glycolysis intermediate Glc-6-P; however, its proficiency [kuncat/(kcat/KM)] for SQ was 17 000-fold better than for Glc-6-P, revealing that HsSQM preferentially stabilizes the SQ transition state.

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© 2018, The Author(s).

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