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

Abayakoon, Palika, Lingford, James P., Jin, Yi ORCID:, Bengt, Christopher, Davies, Gideon J., Yao, Shenggen, Goddard-Borger, Ethan D. and Williams, Spencer J. 2018. Discovery and characterization of a sulfoquinovose mutarotase using kinetic analysis at equilibrium by exchange spectroscopy. Biochemical Journal 475 (7) , pp. 1371-1383. 10.1042/BCJ20170947

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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 Herbaspirillum seropedicaea ( 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 ( k cat/ K M) for SQ versus the glycolysis intermediate glucose-6-phosphate (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 stabilises the SQ transition state.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Chemistry
Publisher: Biochemical Society
ISSN: 0264-6021
Date of First Compliant Deposit: 27 March 2018
Date of Acceptance: 13 March 2018
Last Modified: 06 Jan 2024 20:49

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