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.

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.

Inhibitor design incorporating features of the reaction coordinate and transition-state structure has emerged as a powerful approach for the development of enzyme inhibitors. Such inhibitors find use as mechanistic probes, chemical biology tools, and therapeutics. Endo-α-1,2-mannosidases and endo-α-1,2-mannanases, members of glycoside hydrolase family 99 (GH99), are interesting targets for inhibitor development as they play key roles in N-glycan maturation and microbiotal yeast mannan degradation, respectively. These enzymes are proposed to act via a 1,2-anhydrosugar “epoxide” mechanism that proceeds through an unusual conformational itinerary. Here, we explore how shape and charge contribute to binding of diverse inhibitors of these enzymes. We report the synthesis of neutral dideoxy, glucal and cyclohexenyl disaccharide inhibitors, their binding to GH99 endo-α-1,2-mannanases, and their structural analysis by X-ray crystallography. Quantum mechanical calculations of the free energy landscapes reveal how the neutral inhibitors provide shape but not charge mimicry of the proposed intermediate and transition state structures. Building upon the knowledge of shape and charge contributions to inhibition of family GH99 enzymes, we design and synthesize α-Man-1,3-noeuromycin, which is revealed to be the most potent inhibitor (KD 13 nM for Bacteroides xylanisolvens GH99 enzyme) of these enzymes yet reported. This work reveals how shape and charge mimicry of transition state features can enable the rational design of potent inhibitors.

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.

The enzymatic cleavage of β-1,4-mannans is achieved by endo-β-1,4-mannanases, enzymes involved in germination of seeds and microbial hemicellulose degradation, and which have increasing industrial and consumer product applications. β-Mannanases occur in a range of families of the CAZy sequence-based glycoside hydrolase (GH) classification scheme including families 5, 26, and 113. In this work we reveal that β-mannanases of the newly described GH family 134 differ from other mannanase families in both their mechanism and tertiary structure. A representative GH family 134 endo-β-1,4-mannanase from a Streptomyces sp. displays a fold closely related to that of hen egg white lysozyme but acts with inversion of stereochemistry. A Michaelis complex with mannopentaose, and a product complex with mannotriose, reveal ligands with pyranose rings distorted in an unusual inverted chair conformation. Ab initio quantum mechanics/molecular mechanics metadynamics quantified the energetically accessible ring conformations and provided evidence in support of a 1C4 → 3H4‡ → 3S1 conformational itinerary along the reaction coordinate. This work, in concert with that on GH family 124 cellulases, reveals how the lysozyme fold can be co-opted to catalyze the hydrolysis of different polysaccharides in a mechanistically distinct manner.

Bacteroides vulgatus is a member of the human microbiota whose abundance is increased in patients with Crohn's disease. We show that a B. vulgatus glycoside hydrolase from the carbohydrate active enzyme family GH123, BvGH123, is an N-acetyl-β-galactosaminidase that acts with retention of stereochemistry, and, through a 3-D structure in complex with Gal-thiazoline, provide evidence in support of a neighbouring group participation mechanism.

Catabolism of sulfoquinovose (SQ,, the ubiquitous sulfosugar produced by photosynthetic organisms, is an important component of the biogeochemical carbon and sulfur cycles.Here, we describe a new 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 (SQGro) are imported into the cell by an ABC transporter system with an associated SQ binding protein. A sulfoquinovosidase hydrolyses the SQ glycoside and the liberated SQ is acted on by a flavin mononucleotide-dependent sulfoquinovose monooxygenase, in concert with an NADHdependent flavin reductase, to release sulfite and 6-oxo-glucose. An NADPH-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 (smo) 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 as it enables the complete utilization of all carbons within SQ by a single organism with concomitant production of inorganic sulfite. Significance StatementSulfoquinovose, a sulfosugar derivative of glucose, is produced by most photosynthetic organisms and contains up to half of all sulfur in the biosphere. Several pathways for its breakdown are known, though they provide access to only half of the carbon in sulfoquinovose and none of its sulfur. Here, we describe a fundamentally different pathway within the plant pathogen Agrobacterium tumefaciens that features oxidative desulfurization of sulfoquinovose to access all carbon and sulfur within the molecule. Biochemical and structural analyses of the pathway's key proteins provided insights how the sulfosugar is recognized and degraded. Genes encoding this sulfoquinovose monooxygenase pathway are present in many plant pathogens and symbionts, alluding to a possible role for sulfoquinovose in plant host-bacteria interactions.

Bacteroides vulgatus is a member of the human microbiota whose abundance is increased in patients with Crohn's disease. We show that a B. vulgatus glycoside hydrolase from the carbohydrate active enzyme family GH123, BvGH123, is an N-acetyl-β-galactosaminidase that acts with retention of stereochemistry, and through a 3-D structure in complex with Gal-thiazoline, provide evidence in support of a neighbouring group participation mechanism.