The Molecular Basis of Sulfosugar Selectivity in Sulfoglycolysis

The sulfosugar sulfoquinovose (SQ) is produced by essentially all photosynthetic organisms on Earth and is metabolized by bacteria through the process of sulfoglycolysis. The sulfoglycolytic Embden-Meyerhof-Parnas pathway metabolizes 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 (SFP) aldolase. Our data show 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 three-dimensional structures of each enzyme, which reveal the presence of conserved sulfonate binding pockets. We show that SQ isomerase acts preferentially on the β-anomer of SQ and reversibly produces both SF and sulforhamnose (SR), a previously unknown sugar that acts as a derepressor for the transcriptional repressor CsqR that regulates SQ-utilization. We also demonstrate that SF kinase is a key regulatory enzyme for the pathway that experiences complex modulation by the metabolites SQ, SLA, AMP, ADP, ATP, F6P, FBP, PEP, DHAP, and citrate, and we show that SFP aldolase reversibly synthesizes SFP. 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

Structural and biochemical insights into the function and evolution of sulfoquinovosidases

An estimated 10 billion tonnes of sulfoquinovose (SQ) are produced and degraded each year. Prokaryotic sulfoglycolytic pathways catabolize sulfoquinovose (SQ) liberated from plant sulfolipid, or its delipidated form α-d-sulfoquinovosyl glycerol (SQGro), through the action of a sulfoquinovosidase (SQase), but little is known about the capacity of SQ glycosides to support growth. Structural studies of the first reported SQase (Escherichia coli YihQ) have identified three conserved residues that are essential for substrate recognition, but crossover mutations exploring active-site residues of predicted SQases from other organisms have yielded inactive mutants casting doubt on bioinformatic functional assignment. Here, we show that SQGro can support the growth of E. coli on par with d-glucose, and that the E. coli SQase prefers the naturally occurring diastereomer of SQGro. A predicted, but divergent, SQase from Agrobacterium tumefaciens proved to have highly specific activity toward SQ glycosides, and structural, mutagenic, and bioinformatic analyses revealed the molecular coevolution of catalytically important amino acid pairs directly involved in substrate recognition, as well as structurally important pairs distal to the active site. Understanding the defining features of SQases empowers bioinformatic approaches for mapping sulfur metabolism in diverse microbial communities and sheds light on this poorly understood arm of the biosulfur cycle

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

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

Development of a molecular description of the Embden-Meyerhof-Parnas sulfoglycolysis pathway

© 2019 Palika AbayakoonSulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) is the polar head group of the plant sulfolipid, sulfoquinovoside diacylglycerol (SQDG). SQ is estimated to be synthesized at a scale of some 10 billion tonnes annually and is a major contributor to the biogeochemical sulfur cycle. SQDG is a thylakoid membrane sulfolipid and is involved in membrane structure and function, and modulates the activity of photosynthetic complexes. Two sulfoglycolysis pathways have been discovered in recent years that allow the degradation of SQDG: the sulfoglycolytic Embden-Meyerhof-Parnas (sulfo-EMP) pathway and the sulfoglycolytic Entner-Doudoroff (sulfo-ED) pathway. SQDG hydrolysis to form SQ, enabling entry of SQDG to the sulfur cycle, is catalyzed by glycosidases termed sulfoquinovosidases (SQases). Structural analysis of the first SQase, EcYihQ from Escherichia coli, revealed substrate recognition by a QQRWY motif in the active site. Other putative sulfoquinovosidases contain a KERWY motif; it is not known whether they are genuine sulfoquinovosidases. In Chapter 2 we present the discovery and characterization of a second sulfoquinovosidase that bears a KERWY motif, AtSQase from Agrobacterium tumefaciens. AtSQase catalyzes hydrolysis of the artificial substrate p-nitrophenyl sulfoquinovoside (PNPSQ), which enabled its kinetic and structural characterization. Through the synthesis of a series of analogues of PNPSQ it is shown that EcYihQ and AtSQase are highly specific for both correct substrate stereochemistry and the sulfonate group. The first SQase inhibitor SQ-IFG was designed and synthesized. Structural analysis of both enzymes allowed the identification of catalytic and substrate binding residues. Their roles were supported by mutagenesis and kinetic analysis. Mutual information analysis provided insights into the evolution of these proteins. Chapter 3 covers studies of sulfoquinovose mutarotase activity using a homolog of the putative mutarotase YihR from E. coli, namely the sulfoquinovose mutarotase from Herbaspirillum seropedicae (HsSQM). With the use of 1D and 2D EXSY NMR techniques, unidirectional mutarotation rates in equilibrium mixtures of various hexoses, including SQ were measured. The enzyme exhibited a broad spectrum mutarotase activity but did not tolerate an axial C2 hydroxyl group. Further studies demonstrated that this enzyme is a sulfoquinovose mutarotase with approximately 17 000-fold preference for SQ compared to glucose 6-phosphate. Chapter 4 explored the catalytic activity of E. coli YihS catalyzing isomerization of SQ and 6-deoxy-6-sulfofructose (SF). Both substrates for the enzyme were synthesized, and NMR studies demonstrated the reversible interconversion of SQ to SF with formation of a new product, sulforhamnose (SR, 6-deoxy-6-sulfomannose). HPLC analysis showed that EcYihS catalysed the isomerisation of SQ at a rate approximately 178-fold greater compared to D-mannose (a previously described substrate for this enzyme), in terms of kcat/KM. NMR studies of the rate of YihS catalyzed H/D isotope exchange revealed that EcYihS prefers beta-SQ as the substrate. Chapter 5 of the thesis focused on the EcYihU catalyzed reduction of sulfolactaldehyde (SLA) to dihydroxypropane-1-sulfonate (DHPS). The substrate SLA was synthesized and its stability was defined. By monitoring consumption of cofactor, the rate of EcYihU catalyzed conversion of SLA to DHPS was measured, showing higher activity compared to succinic semialdehyde, a previously described substrate for this enzyme. Reduced forms of the cofactor NADH were synthesized; analysis of their inhibitory potency revealed that tetrahydro-NADH was more potent than hexahydro-NADH. Mechanistic studies using these inhibitors supported a sequential kinetic mechanism for EcYihU. X-ray studies have identified the sulfonate binding residues and revealed domain movements in YihU upon substrate/cofactor binding

Dynamic Structural Changes Accompany the Production of 2-Dihydroxypropanesulfonate by Sulfolactaldehyde Reductase

2,3-Dihydroxypropanesulfonate (DHPS) is a major sulfur species in the biosphere. One important route for the production of DHPS includes 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. On the basis of conserved sequence motifs, we assign SLA reductase to the β-hydroxyacid dehydrogenase (β-HAD) family, making it the first example of a β-HAD enzyme that acts on a sulfonic acid, rather than a carboxylic acid substrate. We report crystal structures of the SLA reductase YihU from E. coli K-12 in its apo and cofactor-bound states, as well as the 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 unique peptide sequence that is predictive of SLA reductase activity.<br /

The Molecular Basis of Sulfosugar Selectivity in Sulfoglycolysis

The sulfosugar sulfoquinovose (SQ) is produced by essentially all photosynthetic organisms on earth and is metabolized by 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 (SFP) 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 enzyme, which reveal the presence of conserved sulfonate-binding pockets. We show that SQ isomerase acts preferentially on the β-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-utilization. We also demonstrate that SF kinase is a key regulatory enzyme for the pathway that experiences complex modulation by the metabolites AMP, ADP, ATP, F6P, FBP, PEP, and citrate, and we show that SFP aldolase reversibly synthesizes SFP. 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