Comprehensive Synthesis of Substrates, Intermediates, and Products of the Sulfoglycolytic Embden–Meyerhoff–Parnas Pathway

Abayakoon, Palika; Epa, Ruwan; Petricevic, Marija; Bengt, Christopher; Mui, Janice; Peet, Phillip L. van der; Zhang, Yunyang; Lingford, James P.; White, Jonathan M.; Goddard‐Borger, Ethan D.; Williams, Spencer J.

doi:10.1021/acs.joc.9b00055

“…Sulfogluconate (SG) was synthesized by oxidation of SQ with iodine 25 (Figures S11 and S12), while SQ, SF, SFP, DHPS, SLA and SL were prepared as previously reported. 26 Rl -SRDI565 was grown to mid-log phase in Y 5% M or Y 5% SQ, metabolically quenched and extracted polar metabolites analyzed by LC/MS-MS. SQ-grown bacteria contained SQ, SF, SG, SL and DHPS, while SFP and SLA could not be detected (Figures 5a-e, Figure S14). The detection of SG is characteristic for a sulfo-ED pathway, and presumably arises from the action of the putative SQ dehydrogenase and SGL lactonase.…”

Section: Resultsmentioning

confidence: 99%

“…SQ, ( 13 C 6 )-SQ, SF, SFP, SLA, SL, and DHPS were synthesized as described previously. 26 IFG-SQ was prepared as described. 24…”

Section: Methodsmentioning

confidence: 99%

A sulfoglycolytic Entner-Doudoroff pathway inRhizobium leguminosarumbv.trifoliiSRDI565

Li¹,

Epa²,

Scott

³

et al. 2019

Preprint

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32Rhizobia are nitrogen fixing bacteria that engage in symbiotic relationships with plant hosts 33 but can also persist as free-living bacteria with the soil and rhizosphere. Here we show that free 34 living Rhizobium leguminosarum SRDI565 can grow on the sulfosugar sulfoquinovose (SQ) 35 using a sulfoglycolytic Entner-Doudoroff (sulfo-ED) pathway resulting in production of 36 sulfolactate (SL) as the major metabolic end-product. Comparative proteomics supports the 37 involvement of a sulfo-ED operon encoding an ABC transporter cassette, sulfo-ED enzymes 38 and an SL exporter. Consistent with an oligotrophic lifestyle, proteomics data revealed little 39 change in expression of the sulfo-ED proteins during growth on SQ versus mannitol, a result 40 confirmed through biochemical assay of sulfoquinovosidase activity in cell lysates (data are 41 available via ProteomeXchange with identifier PXD015822). Metabolomics analysis showed 42 that growth on SQ involves gluconeogenesis to satisfy metabolic requirements for glucose-6-43 phosphate and fructose-6-phosphate. Metabolomics analysis also revealed the unexpected 44 production of small amounts of sulfofructose and 2,3-dihydroxypropanesulfonate, which are 45 proposed to arise from promiscuous activities of the glycolytic enzyme phosphoglucose 46 isomerase and a non-specific aldehyde reductase, respectively. This work shows that rhizobial 47 metabolism of the abundant sulfosugar SQ may contribute to persistence of the bacteria in the 48 soil and to mobilization of sulfur in the pedosphere. 49 50 Results 127Analysis of the genome of Rl-SRDI565 revealed a sulfo-ED operon that had the same 128 genes, but no synteny with the P. putida SQ1 operon (Figure 1). Genes with high homology to 129 the P. putida proteins included a putative SQase, SQ dehydrogenase, SL lactonase, SG 130 dehydratase, KDSG aldolase and SLA dehydrogenase, and an SL exporter (see Figures S1-S6). 131The Rl-SRDI565 operon contains some important differences compared to that of P. putida 132 SQ1. In particular, it lacks a putative SQ mutarotase, 20 and appears to use an ABC transporter 133 cassette to import SQ/SQGro in place of an SQ/SQGro importer/permease. The putative sulfo-134 ED pathway in Rl-SRDI565 is consistent with the proposed protein functions outlined in Figure 135 1b, with a comparison to the classical ED pathway in Figure 1c. 136

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“…Recombinantly expressed YihT from E. coli was incubated with chemo-enzymatically synthesized SFP; 18 however, we were unable to detect the formation of DHAP or SLA under a range of conditions. Instead, we recombinantly expressed YihT from Salmonella enterica, which shares 87% similarity) ( Figure S17).…”

Section: Sfp Aldolasementioning

confidence: 67%

The Molecular Basis of Sulfosugar Selectivity in Sulfoglycolysis

Sharma¹,

Abayakoon²,

Jiang³

et al. 2020

Preprint

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The sulfosugar sulfoquinovose (SQ) is produced by essentially all photosynthetic organisms on earth and is metabolized bacteria through the process of sulfoglycolysis. The sulfoglycolytic Embden-Meyerhof-Parnas pathway metabolises SQ to produce dihydroxyacetone phosphate and sulfolactaldehyde and is analogous to the classical Embden-Meyerhof-Parnas glycolysis pathway for the metabolism of glucose-6-phosphate, though the former only provides one C3 fragment to central metabolism, with excretion of the other C3 fragment as dihydroxypropanesulfonate. Here, we report a comprehensive structural and biochemical analysis of the three core steps of sulfoglycolysis catalyzed by SQ isomerase, sulfofructose (SF) kinase and sulfofructose-1-phosphate aldolase. Our data shows that despite the superficial similarity of this pathway to glycolysis, the sulfoglycolytic enzymes are specific for SQ metabolites and are not catalytically active on related metabolites from glycolytic pathways. This observation is rationalized by 3D structures of each of these enzymes, which reveal the presence of conserved sulfonate-binding pockets. We show that SQ isomerase acts preferentially on the b-anomer of SQ and reversibly produces both SF and sulforhamnose (SR), a previously unknown sugar that acts as a transcriptional regulator for the transcriptional repressor CsqR that regulates SQ-utilisation. We also demonstrate that SF kinase is a key regulatory enzyme for the pathway that experiences complex allosteric modulation by the metabolites AMP, ADP, ATP, F6P, FBP, PEP, and citrate. This body of work provides fresh insights into the mechanism, specificity and regulation of sulfoglycolysis and has important implications for understanding how this biochemistry interfaces with central metabolism in prokaryotes to process this major repository of biogeochemical sulfur.

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“…One product was identified as SF through comparison with authentic material. 18 Resonances for the second new compound did not match either anomer of SQ or SF, particularly singlets in the 1 H NMR spectrum at  4.86 and 5.10 ppm, and in the 13 C NMR spectrum at  74.6 and 71.7 ppm. Further insight into the structure of the unknown product was obtained by conducting an additional experiment on the reaction of SQ with EcYihS, this time in buffered D2O.…”

Section: Sq-sf Isomerase Also Produces Sulforhamnosementioning

confidence: 86%

“…We established an HPLC-MS/MS assay for EcYihV ( Figure S8) using a ZIC-HILIC column and chemically synthesized SF substrate ( Figure S9). 18 Initially, we measured the kinetics for production of SFP. To delineate the specific interactions with substrates and products, three crystal structures of EcYihV were obtained in complex with substrate SF and AMPPNP or ADP as ATP analogues, as well as product SFP.…”

Section: Sf Kinase Is Allosterically Regulated By Central Metabolitesmentioning

confidence: 99%

The Molecular Basis of Sulfosugar Selectivity in Sulfoglycolysis

Sharma¹,

Abayakoon²,

Epa³

et al. 2021

Preprint

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0

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The sulfosugar sulfoquinovose (SQ) is produced by essentially all photosynthetic organisms on earth and is metabolized bacteria through the process of sulfoglycolysis. The sulfoglycolytic Embden-Meyerhof-Parnas pathway metabolises SQ to produce dihydroxyacetone phosphate and sulfolactaldehyde and is analogous to the classical Embden-Meyerhof-Parnas glycolysis pathway for the metabolism of glucose-6-phosphate, though the former only provides one C3 fragment to central metabolism, with excretion of the other C3 fragment as dihydroxypropanesulfonate. Here, we report a comprehensive structural and biochemical analysis of the three core steps of sulfoglycolysis catalyzed by SQ isomerase, sulfofructose (SF) kinase and sulfofructose-1-phosphate aldolase. Our data shows that despite the superficial similarity of this pathway to glycolysis, the sulfoglycolytic enzymes are specific for SQ metabolites and are not catalytically active on related metabolites from glycolytic pathways. This observation is rationalized by 3D structures of each of these enzymes, which reveal the presence of conserved sulfonate-binding pockets. We show that SQ isomerase acts preferentially on the b-anomer of SQ and reversibly produces both SF and sulforhamnose (SR), a previously unknown sugar that acts as a transcriptional regulator for the transcriptional repressor CsqR that regulates SQ-utilisation. We also demonstrate that SF kinase is a key regulatory enzyme for the pathway that experiences complex allosteric modulation by the metabolites AMP, ADP, ATP, F6P, FBP, PEP, and citrate. This body of work provides fresh insights into the mechanism, specificity and regulation of sulfoglycolysis and has important implications for understanding how this biochemistry interfaces with central metabolism in prokaryotes to process this major repository of biogeochemical sulfur.

show abstract

Comprehensive Synthesis of Substrates, Intermediates, and Products of the Sulfoglycolytic Embden–Meyerhoff–Parnas Pathway

Cited by 35 publications

References 41 publications

A sulfoglycolytic Entner-Doudoroff pathway inRhizobium leguminosarumbv.trifoliiSRDI565

A sulfoglycolytic Entner-Doudoroff pathway inRhizobium leguminosarumbv.trifoliiSRDI565

The Molecular Basis of Sulfosugar Selectivity in Sulfoglycolysis

The Molecular Basis of Sulfosugar Selectivity in Sulfoglycolysis

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