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Abstract: 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.

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Abstract: Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 endo-α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope (E3) conformation. Kinetic isotope effects (kcat/KM) for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.

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Abstract: 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.

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Abstract: Understanding the origins of lipid membrane bilayer rearrangement in response to external stimuli is an essential component of cell biology and the bottom-up design of liposomes for biomedical applications. The enzymes phospholipase C and D (PLC and PLD) both cleave the phosphorus–oxygen bonds of phosphate esters in phosphatidylcholine (PC) lipids. The atomic position of this hydrolysis reaction has huge implications for the stability of PC-containing self-assembled structures, such as the cell wall and lipid-based vesicle drug delivery vectors. While PLC converts PC to diacylglycerol (DAG), the interaction of PC with PLD produces phosphatidic acid (PA). Here we present a combination of small-angle scattering data and all-atom molecular dynamics simulations, providing insights into the effects of atomic-scale reorganization on the supramolecular assembly of PC membrane bilayers upon enzyme-mediated incorporation of DAG or PA. We observed that PC liposomes completely disintegrate in the presence of PLC, as conversion of PC to DAG progresses. At lower concentrations, DAG molecules within fluid PC bilayers form hydrogen bonds with backbone carbonyl oxygens in neighboring PC molecules and burrow into the hydrophobic region. This leads initially to membrane thinning followed by a swelling of the lamellar phase with increased DAG. At higher DAG concentrations, localized membrane tension causes a change in lipid phase from lamellar to the hexagonal and micellar cubic phases. Molecular dynamics simulations show that this destabilization is also caused in part by the decreased ability of DAG-containing PC membranes to coordinate sodium ions. Conversely, PLD-treated PC liposomes remain stable up to extremely high conversions to PA. Here, the negatively charged PA headgroup attracts significant amounts of sodium ions from the bulk solution to the membrane surface, leading to a swelling of the coordinated water layer. These findings are a vital step toward a fundamental understanding of the degradation behavior of PC lipid membranes in the presence of these clinically relevant enzymes, and toward the rational design of diagnostic and drug delivery technologies for phospholipase-dysregulation-based diseases.

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Abstract: The physical properties of 3-D porous solids are defined by their molecular geometry. Hence, precise control of pore size, pore shape, and pore connectivity are needed to tailor them for specific applications. However, for porous molecular crystals, the modification of pore size by adding pore-blocking groups can also affect crystal packing in an unpredictable way. This precludes strategies adopted for isoreticular metal–organic frameworks, where addition of a small group, such as a methyl group, does not affect the basic framework topology. Here, we narrow the pore size of a cage molecule, CC3, in a systematic way by introducing methyl groups into the cage windows. Computational crystal structure prediction was used to anticipate the packing preferences of two homochiral methylated cages, CC14-R and CC15-R, and to assess the structure–energy landscape of a CC15-R/CC3-S cocrystal, designed such that both component cages could be directed to pack with a 3-D, interconnected pore structure. The experimental gas sorption properties of these three cage systems agree well with physical properties predicted by computational energy–structure–function maps.