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

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

doi:10.1042/bcj20170947

Palika Abayakoon, Ethan D Goddard-Borger + Show 6 more

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https://doi.org/10.1042/bcj20170947

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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.

Highlights

Various prokaryotes metabolize the sugar sulfoquinovose (SQ) to sulfolactaldehyde (SLA) and dihydroxyacetone phosphate (DHAP), via an Embden–Meyerhof–Parnas (EMP)-like pathway [1], or pyruvate, via an Entner–Doudoroff (ED)-like pathway [2,3] (Figure 1)
SQ is rarely encountered as a free sugar in nature; rather, it is liberated from the plant sulfolipid α-sulfoquinovosyl diacylglycerol (SQDG), or its delipidated form α-sulfoquinovosyl glycerol (SQGro), by the action of glycoside hydrolases termed sulfoquinovosidases (SQases) [4]
We turned our attention to other putative SQ mutarotases from various bacteria that possess sulfoglycolytic gene clusters and succeeded in obtaining WP_069374721.1 from H. seropedicaea in high yield and purity

Summary

Introduction

Various prokaryotes metabolize the sugar sulfoquinovose (SQ) to sulfolactaldehyde (SLA) and dihydroxyacetone phosphate (DHAP), via an Embden–Meyerhof–Parnas (EMP)-like pathway [1], or pyruvate, via an Entner–Doudoroff (ED)-like pathway [2,3] (Figure 1). All proteins comprising a sulfo-ED or sulfo-EMP pathway are typically encoded within a single gene cluster. These clusters usually include an SQase, which highlights that SQ glycosides are important natural feedstocks for sulfoglycolytic pathways [1,2]. The gene clusters encode a conserved uncharacterized protein, annotated as an aldose-1-epimerase, which likely catalyzes SQ mutarotation: an enzyme activity yet to be reported. Mutarotases are widely distributed enzymes that facilitate the rapid mutarotation of aldoses to enhance flux through metabolic pathways when enzymes acting on reducing sugars are specific for a single anomer [5,6]

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