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

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 (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 HsSQM preferentially stabilizes the SQ transition state.

Keywords: NMR spectroscopy; enzymology; metabolism; mutarotase; sulfoglycolysis.

Figure 1.. Summary of sulfoglycolysis.

Importation of SQGro and cleavage by SQase, or direct importation of SQ, provides an intracellular pool of SQ anomers that can be interconverted by SQ mutarotase. SQ is metabolized by sulfo-ED or sulfo-EMP pathways to SLA and then to the C₃-sulfonates DHPS or SL, prior to export.

Figure 2.. H. seropedicaea contains a sulfo-ED operon and a putative SQ mutarotase.

(A) Operon structure of P. putida SQ1 and H. seropedicaea strain AU14040. Bold indicates genes for which enzymatic activity has been biochemically determined in at least one organism. (B) Alignment of various putative mutarotases with secondary structural elements. WP_069374721.1, HsQM from H. seropedicaea; NP_418315.3, YihQ from E. coli; KHL76357.1, PpSQ1_00415 from Pseudomonas putida SQ1; DAA09996.1, YMR099C hexose-1-phosphate mutarotase from S. cerevisiae; BAE76730.1, YphB aldose-1-epimerase from E. coli (BAE76730.1). The secondary structural elements are annotated from the structure of E. coli YphB (PDB 3nre).

Figure 3.. Homology model of HsSQM.

(A) Overlay of HsSQM homology model (green) with a putative mutarotase from C. acetobutylicum (PDB 3OS7; gray). (B) The active sites of the HsSQM homology model (green) overlaid with the galactose mutarotase domain of gal10 from S. cerevisae with d-galactose bound (gray, PDB 1Z45). Residue numbers are for HsSQM.

Figure 4.. Excerpt showing anomeric regions of 2D ¹H-¹H EXSY plots of various hexoses alone and with H. seropediacae mutarotase.

(A) Sulfoquinovose (SQ), (B) d-glucose-6-phosphate (Glc-6-P), (C) d-glucuronic acid (GlcA), (D) d-glucose (Glc), (E) d-galactose (Gal), and (F) d-mannose (Man). Hexoses are at 5 mM, 1.51 μM HsSQM in 50 mM sodium phosphate, 150 mM NaCl (pD 7.5).

Figure 5.. Kinetic analysis of HsSQM by inversion recovery 1D ¹H EXSY.

Inversion recovery curves for 10 mM SQ or Glc-6-P corresponding to (A) α-SQ at 4.00 mM and (B) α-Glc-6-P at 3.57 mM. Inversion decay curves corresponding to (C) β-SQ at 6.00 mM and (D) β-Glc-6-P at 6.43 mM. Michaelis–Menten plots for (E) β-SQ and (F) β-Glc-6-P. Dashed lines indicate tangents to the fitted curve at t = 0.

Figure 6.. Spontaneous mutarotation of α-SQ and pD dependence of HsSQM.

(A) Plot of rates of spontaneous mutarotation of α-SQ versus phosphate buffer concentration at pseudo-constant ionic strength. Buffers consisted of 10–50 mM sodium phosphate and 2 M NaCl in D₂O (pD 7.5). For comparison, k = 0.0073 min⁻¹ in 50 mM sodium phosphate and 150 mM NaCl in D₂O (pD 7.5). (B) pD dependence of HsSQM activity for mutarotation of Glc-6-P. Data were fit to a bell-shaped curve leading to estimated pK_a values of 5.9 ± 0.1 and 9.9 ± 0.1.