No abstract

2,3-Dihydroxypropanesulfonate (DHPS) is a major sulfur species in the biosphere. One important route for the production of DHPS is sulfoglycolytic catabolism of sulfoquinovose (SQ) through the Embden-Meyerhof-Parnas (sulfo-EMP) pathway. SQ is a sulfonated carbohydrate present in plant and cyanobacterial sulfolipids (sulfoquinovosyl diacylglyceride and its metabolites) and is biosynthesised globally at a rate of around 10 billion tonnes per annum. The final step in the bacterial sulfo-EMP pathway involves reduction of sulfolactaldehyde (SLA) to DHPS, catalysed by an NADH-dependent SLA reductase. Based on conserved sequence motifs, we assign SLA reductase to the β-hydroxyacid dehydrogenase (β-HAD) family, an example of a β-HAD enzyme that acts on a sulfonic acid substrate, rather than a carboxylic acid. We report crystal structures of the SLA reductase YihU from E. coli K-12 in its apo and cofactor-bound states, as well as a ternary complex YihU•NADH•DHPS with the cofactor and product bound in the active site. Conformational flexibility observed in these structures, combined with kinetic studies, confirm a sequential mechanism and provide evidence for dynamic domain movements that occur during catalysis. The ternary complex structure reveals a conserved sulfonate pocket in SLA reductase that recognises the sulfonate oxygens through hydrogen bonding to Asn174, Ser178, and the backbone amide of Arg123, along with an ordered water molecule. This triad of residues distinguishes these enzymes from classical β-HADs that act on carboxylate substrates. A comparison of YihU crystal structures with close structural homologues within the β-HAD family highlights key differences in the overall domain organization and identifies a peptide sequence that is predictive of SLA reductase activity.

Sulfoglycolysis is a metabolic pathway dedicated to the catabolism of the sulfosugar sulfoquinovose (SQ) into smaller organosulfur fragments. An estimated 10 billion tonnes of SQ fluxes through sulfoglycolysis pathways each year, making it a significant aspect of the biogeochemical sulfur cycle. Delineating the molecular details of sulfoglycolysis requires authentic samples of the various metabolites in these pathways. To this end, we have established chemical and chemoenzymatic methods for the synthesis of the key organosulfur metabolites sulfoquinovosylglycerol, SQ (also in 13C6-labeled form), sulfofructose, sulfofructose-1-phosphate, sulfolactaldehyde, and 2,3-dihydroxypropanesulfonate, as well as an improved route to the chromogenic sulfoquinovosidase substrate 4-nitrophenyl α-sulfoquinovoside.

Rhizobia are nitrogen-fixing bacteria that engage in symbiotic relationships with plant hosts but can also persist as free-living bacteria in the soil and rhizosphere. Here, we show that free-living Rhizobium leguminosarum SRDI565 can grow on the sulfosugar sulfoquinovose (SQ) or the related glycoside SQ-glycerol using a sulfoglycolytic Entner-Doudoroff (sulfo-ED) pathway, resulting in production of sulfolactate (SL) as the major metabolic end product. Comparative proteomics supports the involvement of a sulfo-ED operon encoding an ABC transporter, sulfo-ED enzymes, and an SL exporter. Consistent with an oligotrophic lifestyle, proteomics data revealed little change in expression of the sulfo-ED proteins during growth on SQ versus mannitol, a result confirmed through biochemical assay of sulfoquinovosidase activity in cell lysates. Metabolomics analysis showed that growth on SQ involves gluconeogenesis to satisfy metabolic requirements for glucose-6-phosphate and fructose-6-phosphate. Metabolomics analysis also revealed the unexpected production of small amounts of sulfofructose and 2,3-dihydroxypropanesulfonate, which are proposed to arise from promiscuous activities of the glycolytic enzyme phosphoglucose isomerase and a nonspecific aldehyde reductase, respectively. The discovery of a rhizobium isolate with the ability to degrade SQ builds our knowledge of how these important symbiotic bacteria persist within soil. IMPORTANCE Sulfonate sulfur is a major form of organic sulfur in soils but requires biomineralization before it can be utilized by plants. Very little is known about the biochemical processes used to mobilize sulfonate sulfur. We show that a rhizobial isolate from soil, Rhizobium leguminosarum SRDI565, possesses the ability to degrade the abundant phototroph-derived carbohydrate sulfonate SQ through a sulfoglycolytic Entner-Doudoroff pathway. Proteomics and metabolomics demonstrated the utilization of this pathway during growth on SQ and provided evidence for gluconeogenesis. Unexpectedly, off-cycle sulfoglycolytic species were also detected, pointing to the complexity of metabolic processes within cells under conditions of sulfoglycolysis. Thus, rhizobial metabolism of the abundant sulfosugar SQ may contribute to persistence of the bacteria in the soil and to mobilization of sulfur in the pedosphere.

Catabolism of sulfoquinovose (SQ; 6-deoxy-6-sulfoglucose), the ubiquitous sulfosugar produced by photosynthetic organisms, is an important component of the biogeochemical carbon and sulfur cycles. Here, we describe a pathway for SQ degradation that involves oxidative desulfurization to release sulfite and enable utilization of the entire carbon skeleton of the sugar to support the growth of the plant pathogen Agrobacterium tumefaciens. SQ or its glycoside sulfoquinovosyl glycerol are imported into the cell by an ATP-binding cassette transporter system with an associated SQ binding protein. A sulfoquinovosidase hydrolyzes the SQ glycoside and the liberated SQ is acted on by a flavin mononucleotide-dependent sulfoquinovose monooxygenase, in concert with an NADH-dependent flavin reductase, to release sulfite and 6-oxo-glucose. An NAD(P)H-dependent oxidoreductase reduces the 6-oxo-glucose to glucose, enabling entry into primary metabolic pathways. Structural and biochemical studies provide detailed insights into the recognition of key metabolites by proteins in this pathway. Bioinformatic analyses reveal that the sulfoquinovose monooxygenase pathway is distributed across Alpha- and Betaproteobacteria and is especially prevalent within the Rhizobiales order. This strategy for SQ catabolism is distinct from previously described pathways because it enables the complete utilization of all carbons within SQ by a single organism with concomitant production of inorganic sulfite.