Capture-and-release of a sulfoquinovose-binding protein on sulfoquinovose-modified agarose.

The solute-binding protein (SBP) components of periplasmic binding protein-dependent ATP-binding cassette (ABC)-type transporters often possess exquisite selectivity for their cognate ligands. Maltose binding protein (MBP), the best studied of these SBPs, has been extensively used as a fusion partner to enable the affinity purification of recombinant proteins. However, other SBPs and SBP-ligand based affinity systems remain underexplored. The sulfoquinovose-binding protein SmoF, is a substrate-binding protein component of the ABC transporter cassette in Agrobacterium tumefaciens involved in importing sulfoquinovose (SQ) and its derivatives for SQ catabolism. Here, we show that SmoF binds with high affinity to the octyl glycoside of SQ (octyl-SQ), demonstrating remarkable tolerance to extension of the anomeric substituent. The 3D X-ray structure of the SmoF·octyl-SQ complex reveals accommodation of the octyl chain, which projects to the protein surface, providing impetus for the synthesis of a linker-equipped SQ-amine using a thiol-ene reaction as a key step, and its conjugation to cyanogen bromide modified agarose. We demonstrate the successful capture and release of SmoF from SQ-agarose resin using SQ as competitive eluant, and selectivity for release versus other organosulfonates. We show that SmoF can be captured and purified from a cell lysate, demonstrating the utility of SQ-agarose in capturing SQ binding proteins from complex mixtures. The present work provides a pathway for development of 'capture-and-release' affinity resins for the discovery and study of SBPs.

Structure and mechanism of sulfofructose transaldolase, a key enzyme in sulfoquinovose metabolism.

Sulfoquinovose (SQ) is a key component of plant sulfolipids (sulfoquinovosyl diacylglycerols) and a major environmental reservoir of biological sulfur. Breakdown of SQ is achieved by bacteria through the pathways of sulfoglycolysis. The sulfoglycolytic sulfofructose transaldolase (sulfo-SFT) pathway is used by gut-resident firmicutes and soil saprophytes. After isomerization of SQ to sulfofructose (SF), the namesake enzyme catalyzes the transaldol reaction of SF transferring dihydroxyacetone to 3C/4C acceptors to give sulfolactaldehyde and fructose-6-phosphate or sedoheptulose-7-phosphate. We report the 3D cryo-EM structure of SF transaldolase from Bacillus megaterium in apo and ligand bound forms, revealing a decameric structure formed from two pentameric rings of the protomer. We demonstrate a covalent "Schiff base" intermediate formed by reaction of SF with Lys89 within a conserved Asp-Lys-Glu catalytic triad and defined by an Arg-Trp-Arg sulfonate recognition triad. The structural characterization of the signature enzyme of the sulfo-SFT pathway provides key insights into molecular recognition of the sulfonate group of sulfosugars.

The sulfoquinovosyl glycerol binding protein SmoF binds and accommodates plant sulfolipids.

Sulfoquinovose (SQ) is the anionic headgroup of the ubiquitous plant sulfolipid, sulfoquinovosyl diacylglycerol (SQDG). SQDG can undergo delipidation to give sulfoquinovosyl glycerol (SQGro) and further glycoside cleavage to give SQ, which can be metabolized through microbial sulfoglycolytic pathways. Exogenous SQDG metabolites are imported into bacteria through membrane spanning transporter proteins. The recently discovered sulfoglycolytic sulfoquinovose monooxygenase (sulfo-SMO) pathway in Agrobacterium tumefaciens features a periplasmic sulfoquinovosyl glycerol binding protein, SmoF, and an ATP-binding cassette (ABC) transporter. Here, we use X-ray crystallography, differential scanning fluorimetry and isothermal titration calorimetry to study SQ glycoside recognition by SmoF. This work reveals that in addition to SQGro, SmoF can also bind SQ, a simple methyl glycoside and even a short-chain SQDG analogue. Molecular recognition of these substrates is achieved through conserved interactions with the SQ-headgroup together with more plastic interactions with the aglycones. This suggests that the solute binding protein of A. tumefaciens, and related SQ-binding proteins from other sulfoglycolytic pathways, can provide their host organisms direct access to most of the SQ metabolites known to be produced by phototrophs.

Oxidative desulfurization pathway for complete catabolism of sulfoquinovose by bacteria.

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.

Sulfoglycolysis: catabolic pathways for metabolism of sulfoquinovose.

Sulfoquinovose (SQ), a derivative of glucose with a C6-sulfonate, is produced by photosynthetic organisms and is the headgroup of the sulfolipid sulfoquinovosyl diacylglycerol. The degradation of SQ allows recycling of its elemental constituents and is important in the global sulfur and carbon biogeochemical cycles. Degradation of SQ by bacteria is achieved through a range of pathways that fall into two main groups. One group involves scission of the 6-carbon skeleton of SQ into two fragments with metabolic utilization of carbons 1-3 and excretion of carbons 4-6 as dihydroxypropanesulfonate or sulfolactate that is biomineralized to sulfite/sulfate by other members of the microbial community. The other involves the complete metabolism of SQ by desulfonylation involving cleavage of the C-S bond to release sulfite and glucose, the latter of which can enter glycolysis. The discovery of sulfoglycolytic pathways has revealed a wide range of novel enzymes and SQ binding proteins. Biochemical and structural characterization of the proteins and enzymes in these pathways have illuminated how the sulfonate group is recognized by Nature's catalysts, supporting bioinformatic annotation of sulfoglycolytic enzymes, and has identified functional and structural relationships with the pathways of glycolysis.

A Sulfoglycolytic Entner-Doudoroff Pathway in Rhizobium leguminosarum bv. trifolii SRDI565.

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.