An atlas of protein-protein interactions across mouse tissues.

Cellular processes arise from the dynamic organization of proteins in networks of physical interactions. Mapping the interactome has therefore been a central objective of high-throughput biology. However, the dynamics of protein interactions across physiological contexts remain poorly understood. Here, we develop a quantitative proteomic approach combining protein correlation profiling with stable isotope labeling of mammals (PCP-SILAM) to map the interactomes of seven mouse tissues. The resulting maps provide a proteome-scale survey of interactome rewiring across mammalian tissues, revealing more than 125,000 unique interactions at a quality comparable to the highest-quality human screens. We identify systematic suppression of cross-talk between the evolutionarily ancient housekeeping interactome and younger, tissue-specific modules. Rewired proteins are tightly regulated by multiple cellular mechanisms and are implicated in disease. Our study opens up new avenues to uncover regulatory mechanisms that shape in vivo interactome responses to physiological and pathophysiological stimuli in mammalian systems.

Dual membrane-spanning anti-sigma factors regulate vesiculation in Bacteroides thetaiotaomicron.

Bacteroidota are abundant members of the human gut microbiota that shape the enteric landscape by modulating host immunity and degrading dietary- and host-derived glycans. These processes are mediated in part by Outer Membrane Vesicles (OMVs). Here, we developed a high-throughput screen to identify genes required for OMV biogenesis and its regulation in Bacteroides thetaiotaomicron (Bt). We identified a family of Dual membrane-spanning anti-sigma factors (Dma) that control OMV biogenesis. We conducted molecular and multiomic analyses to demonstrate that deletion of Dma1, the founding member of the Dma family, modulates OMV production by controlling the activity of the ECF21 family sigma factor, Das1, and its downstream regulon. Dma1 has a previously uncharacterized domain organization that enables Dma1 to span both the inner and outer membrane of Bt. Phylogenetic analyses reveal that this common feature of the Dma family is restricted to the phylum Bacteroidota. This study provides mechanistic insights into the regulation of OMV biogenesis in human gut bacteria.

Ion Mobility-Based Enrichment-Free N-Terminomics Analysis Reveals Novel Legumain Substrates in Murine Spleen.

Aberrant levels of the asparaginyl endopeptidase legumain have been linked to inflammation, neurodegeneration, and cancer, yet our understanding of this protease is incomplete. Systematic attempts to identify legumain substrates have been previously confined to in vitro studies, which fail to mirror physiological conditions and obscure biologically relevant cleavage events. Using high-field asymmetric waveform ion mobility spectrometry (FAIMS), we developed a streamlined approach for proteome and N-terminome analyses without the need for N-termini enrichment. Compared to unfractionated proteomic analysis, we demonstrate FAIMS fractionation improves N-termini identification by >2.5 fold, resulting in the identification of >2882 unique N-termini from limited sample amounts. In murine spleens, this approach identifies 6366 proteins and 2528 unique N-termini, with 235 cleavage events enriched in WT compared to legumain-deficient spleens. Among these, 119 neo-N-termini arose from asparaginyl endopeptidase activities, representing novel putative physiological legumain substrates. The direct cleavage of selected substrates by legumain was confirmed using in vitro assays, providing support for the existence of physiologically relevant extra-lysosomal legumain activity. Combined, these data shed critical light on the functions of legumain and demonstrate the utility of FAIMS as an accessible method to improve depth and quality of N-terminomics studies.

Human gut bacteria tailor extracellular vesicle cargo for the breakdown of diet- and host-derived glycans.

Extracellular vesicles are produced in all three domains of life, and their biogenesis has common ancient origins in eukaryotes and archaea. Although bacterial vesicles were discovered several decades ago and multiple roles have been attributed to them, no mechanism has been established for vesicles biogenesis in bacteria. For this reason, there is a significant level of skepticism about the biological relevance of bacterial vesicles. Bacteroides thetaiotaomicron (Bt), a prominent member of the human intestinal microbiota, produces significant amounts of outer membrane vesicles (OMVs) which have been proposed to play key physiological roles. Here, we employed a dual marker system, consisting of outer membrane- and OMV-specific markers fused to fluorescent proteins to visualize OMV biogenesis by time-lapse microscopy. Furthermore, we performed comparative proteomic analyses to show that, in Bt, the OMV cargo is adapted for the optimal utilization of different polysaccharides. We also show that a negatively charged N-terminal motif acts as a signal for protein sorting into OMVs irrespective of the nutrient availability. Our results demonstrate that OMV production is the result of a highly regulated process in Bt.

Coxiella co-opts the Glutathione Peroxidase 4 to protect the host cell from oxidative stress-induced cell death.

The causative agent of human Q fever, Coxiella burnetii, is highly adapted to infect alveolar macrophages by inhibiting a range of host responses to infection. Despite the clinical and biological importance of this pathogen, the challenges related to genetic manipulation of both C. burnetii and macrophages have limited our knowledge of the mechanisms by which C. burnetii subverts macrophages functions. Here, we used the related bacterium Legionella pneumophila to perform a comprehensive screen of C. burnetii effectors that interfere with innate immune responses and host death using the greater wax moth Galleria mellonella and mouse bone marrow-derived macrophages. We identified MceF (Mitochondrial Coxiella effector protein F), a C. burnetii effector protein that localizes to mitochondria and contributes to host cell survival. MceF was shown to enhance mitochondrial function, delay membrane damage, and decrease mitochondrial ROS production induced by rotenone. Mechanistically, MceF recruits the host antioxidant protein Glutathione Peroxidase 4 (GPX4) to the mitochondria. The protective functions of MceF were absent in primary macrophages lacking GPX4, while overexpression of MceF in human cells protected against oxidative stress-induced cell death. C. burnetii lacking MceF was replication competent in mammalian cells but induced higher mortality in G. mellonella, indicating that MceF modulates the host response to infection. This study reveals an important C. burnetii strategy to subvert macrophage cell death and host immunity and demonstrates that modulation of the host antioxidant system is a viable strategy to promote the success of intracellular bacteria.

Capsular polysaccharide inhibits vaccine-induced O-antigen antibody binding and function across both classical and hypervirulent K2:O1 strains of Klebsiella pneumoniae.

Klebsiella pneumoniae presents as two circulating pathotypes: classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp). Classical isolates are considered urgent threats due to their antibiotic resistance profiles, while hvKp isolates have historically been antibiotic susceptible. Recently, however, increased rates of antibiotic resistance have been observed in both hvKp and cKp, further underscoring the need for preventive and effective immunotherapies. Two distinct surface polysaccharides have gained traction as vaccine candidates against K. pneumoniae: capsular polysaccharide and the O-antigen of lipopolysaccharide. While both targets have practical advantages and disadvantages, it remains unclear which of these antigens included in a vaccine would provide superior protection against matched K. pneumoniae strains. Here, we report the production of two bioconjugate vaccines, one targeting the K2 capsular serotype and the other targeting the O1 O-antigen. Using murine models, we investigated whether these vaccines induced specific antibody responses that recognize K2:O1 K. pneumoniae strains. While each vaccine was immunogenic in mice, both cKp and hvKp strains exhibited decreased O-antibody binding in the presence of capsule. Further, O1 antibodies demonstrated decreased killing in serum bactericidal assays with encapsulated strains, suggesting that the presence of K. pneumoniae capsule blocks O1-antibody binding and function. Finally, the K2 vaccine outperformed the O1 vaccine against both cKp and hvKp in two different murine infection models. These data suggest that capsule-based vaccines may be superior to O-antigen vaccines for targeting hvKp and some cKp strains, due to capsule blocking the O-antigen.

Macrophage infectivity potentiator protein, a peptidyl prolyl cis-trans isomerase, essential for Coxiella burnetii growth and pathogenesis.

Coxiella burnetii is a Gram-negative intracellular pathogen that causes the debilitating disease Q fever, which affects both animals and humans. The only available human vaccine, Q-Vax, is effective but has a high risk of severe adverse reactions, limiting its use as a countermeasure to contain outbreaks. Therefore, it is essential to identify new drug targets to treat this infection. Macrophage infectivity potentiator (Mip) proteins catalyse the folding of proline-containing proteins through their peptidyl prolyl cis-trans isomerase (PPIase) activity and have been shown to play an important role in the virulence of several pathogenic bacteria. To date the role of the Mip protein in C. burnetii pathogenesis has not been investigated. This study demonstrates that CbMip is likely to be an essential protein in C. burnetii. The pipecolic acid derived compounds, SF235 and AN296, which have shown utility in targeting other Mip proteins from pathogenic bacteria, demonstrate inhibitory activities against CbMip. These compounds were found to significantly inhibit intracellular replication of C. burnetii in both HeLa and THP-1 cells. Furthermore, SF235 and AN296 were also found to exhibit antibiotic properties against both the virulent (Phase I) and avirulent (Phase II) forms of C. burnetii Nine Mile Strain in axenic culture. Comparative proteomics, in the presence of AN296, revealed alterations in stress responses with H2O2 sensitivity assays validating that Mip inhibition increases the sensitivity of C. burnetii to oxidative stress. In addition, SF235 and AN296 were effective in vivo and significantly improved the survival of Galleria mellonella infected with C. burnetii. These results suggest that unlike in other bacteria, Mip in C. burnetii is required for replication and that the development of more potent inhibitors against CbMip is warranted and offer potential as novel therapeutics against this pathogen.

Structure, sequon recognition and mechanism of tryptophan C-mannosyltransferase.

C-linked glycosylation is essential for the trafficking, folding and function of secretory and transmembrane proteins involved in cellular communication processes. The tryptophan C-mannosyltransferase (CMT) enzymes that install the modification attach a mannose to the first tryptophan of WxxW/C sequons in nascent polypeptide chains by an unknown mechanism. Here, we report cryogenic-electron microscopy structures of Caenorhabditis elegans CMT in four key states: apo, acceptor peptide-bound, donor-substrate analog-bound and as a trapped ternary complex with both peptide and a donor-substrate mimic bound. The structures indicate how the C-mannosylation sequon is recognized by this CMT and its paralogs, and how sequon binding triggers conformational activation of the donor substrate: a process relevant to all glycosyltransferase C superfamily enzymes. Our structural data further indicate that the CMTs adopt an unprecedented electrophilic aromatic substitution mechanism to enable the C-glycosylation of proteins. These results afford opportunities for understanding human disease and therapeutic targeting of specific CMT paralogs.

Targeting of microvillus protein Eps8 by the NleH effector kinases from enteropathogenic E. coli.

Attaching and effacing (AE) lesion formation on enterocytes by enteropathogenic Escherichia coli (EPEC) requires the EPEC type III secretion system (T3SS). Two T3SS effectors injected into the host cell during infection are the atypical kinases, NleH1 and NleH2. However, the host targets of NleH1 and NleH2 kinase activity during infection have not been reported. Here phosphoproteomics identified Ser775 in the microvillus protein Eps8 as a bona fide target of NleH1 and NleH2 phosphorylation. Both kinases interacted with Eps8 through previously unrecognized, noncanonical "proline-rich" motifs, PxxDY, that bound the Src Homology 3 (SH3) domain of Eps8. Structural analysis of the Eps8 SH3 domain bound to a peptide containing one of the proline-rich motifs from NleH showed that the N-terminal part of the peptide adopts a type II polyproline helix, and its C-terminal "DY" segment makes multiple contacts with the SH3 domain. Ser775 phosphorylation by NleH1 or NleH2 hindered Eps8 bundling activity and drove dispersal of Eps8 from the AE lesion during EPEC infection. This finding suggested that NleH1 and NleH2 altered the cellular localization of Eps8 and the cytoskeletal composition of AE lesions during EPEC infection.

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.

A heptavalent O-antigen bioconjugate vaccine exhibits differential functional antibody responses against diverse Klebsiella pneumoniae isolates.

Klebsiella pneumoniae is the leading cause of neonatal sepsis and is increasingly difficult to treat due to antibiotic resistance. Vaccination represents a tractable approach to combat this resistant bacterium; however, there is currently not a licensed vaccine. Surface polysaccharides, including O-antigens of lipopolysaccharide, have long been attractive candidates for vaccine inclusion. Herein we describe the generation of a bioconjugate vaccine targeting seven predominant O-antigen subtypes in K. pneumoniae. Each bioconjugate was immunogenic in isolation, with limited cross-reactivity among subtypes. Vaccine-induced antibodies demonstrated varying degrees of binding to a wide variety of K. pneumoniae strains. Further, sera from vaccinated mice induced complement-mediated killing of many of these strains. Finally, increased capsule interfered with O-antigen antibodies' ability to bind and mediate killing of some K. pneumoniae strains. Taken together, these data indicate that this novel heptavalent O-antigen bioconjugate vaccine formulation exhibits limited efficacy against some, but not all, K. pneumoniae isolates.

Combining FAIMS based glycoproteomics and DIA proteomics reveals widespread proteome alterations in response to glycosylation occupancy changes in Neisseria gonorrhoeae.

Protein glycosylation is increasingly recognized as a common protein modification across bacterial species. Within the Neisseria genus O-linked protein glycosylation is conserved yet closely related Neisseria species express O-oligosaccharyltransferases (PglOs) with distinct targeting activities. Within this work, we explore the targeting capacity of different PglOs using Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) fractionation and Data-Independent Acquisition (DIA) to allow the characterization of the impact of changes in glycosylation on the proteome of Neisseria gonorrhoeae. We demonstrate FAIMS expands the known glycoproteome of wild type N. gonorrhoeae MS11 and enables differences in glycosylation to be assessed across strains expressing different pglO allelic chimeras with unique substrate targeting activities. Combining glycoproteomic insights with DIA proteomics, we demonstrate that alterations within pglO alleles have widespread impacts on the proteome of N. gonorrhoeae. Examination of peptides known to be targeted by glycosylation using DIA analysis supports alterations in glycosylation occupancy occurs independently of changes in protein levels and that the occupancy of glycosylation is generally low on most glycoproteins. This work thus expands our understanding of the N. gonorrhoeae glycoproteome and the roles that pglO allelic variation may play in governing genus-level protein glycosylation.

Glycan-Tailored Glycoproteomic Analysis Reveals Serine is the Sole Residue Subjected to O-Linked Glycosylation in Acinetobacter baumannii.

Protein glycosylation is a ubiquitous process observed across all domains of life. Within the human pathogen Acinetobacter baumannii, O-linked glycosylation is required for virulence; however, the targets and conservation of glycosylation events remain poorly defined. In this work, we expand our understanding of the breadth and site specificity of glycosylation within A. baumannii by demonstrating the value of strain specific glycan electron-transfer/higher-energy collision dissociation (EThcD) triggering for bacterial glycoproteomics. By coupling tailored EThcD-triggering regimes to complementary glycopeptide enrichment approaches, we assessed the observable glycoproteome of three A. baumannii strains (ATCC19606, BAL062, and D1279779). Combining glycopeptide enrichment techniques including ion mobility (FAIMS), metal oxide affinity chromatography (titanium dioxide), and hydrophilic interaction liquid chromatography (ZIC-HILIC), as well as the use of multiple proteases (trypsin, GluC, pepsin, and thermolysis), we expand the known A. baumannii glycoproteome to 33 unique glycoproteins containing 42 glycosylation sites. We demonstrate that serine is the sole residue subjected to glycosylation with the substitution of serine for threonine abolishing glycosylation in model glycoproteins. An A. baumannii pan-genome built from 576 reference genomes identified that serine glycosylation sites are highly conserved. Combined this work expands our knowledge of the conservation and site specificity of A. baumannii O-linked glycosylation.

Conservation, abundance, glycosylation profile, and localization of the TSP protein family in Cryptosporidium parvum.

Cryptosporidium parvum is a zoonotic apicomplexan parasite and a common cause of diarrheal disease worldwide. The development of vaccines to prevent or limit infection remains an important goal for tackling cryptosporidiosis. At present, the only approved vaccine against any apicomplexan parasite targets a conserved adhesin possessing a thrombospondin repeat domain. C. parvum possesses 12 orthologous thrombospondin repeat domain-containing proteins known as CpTSP1-12, though little is known about these potentially important antigens. Here, we explore the architecture and conservation of the CpTSP protein family, as well as their abundance at the protein level within the sporozoite stage of the life cycle. We examine the glycosylation states of these proteins using a combination of glycopeptide enrichment techniques to demonstrate that these proteins are modified with C-, O-, and N-linked glycans. Using expansion microscopy, and an antibody against the C-linked mannose that is unique to the CpTSP protein family within C. parvum, we show that these proteins are found both on the cell surface and in structures that resemble the secretory pathway of C. parvum sporozoites. Finally, we generated a polyclonal antibody against CpTSP1 to show that it is found at the cell surface and within micronemes, in a pattern reminiscent of other apicomplexan motility-associated adhesins, and is present both in sporozoites and meronts. This work sheds new light on an understudied family of C. parvum proteins that are likely to be important to both parasite biology and the development of vaccines against cryptosporidiosis.

Site-specific immobilization of the endosialidase reveals QSOX2 is a novel polysialylated protein.

Polysialic acid (polySia) is a linear polymer of α2,8-linked sialic acid residues that is of fundamental biological interest due to its pivotal roles in the regulation of the nervous, immune, and reproductive systems in healthy human adults. PolySia is also dysregulated in several chronic diseases, including cancers and mental health disorders. However, the mechanisms underpinning polySia biology in health and disease remain largely unknown. The polySia-specific hydrolase, endoneuraminidase NF (EndoN), and the catalytically inactive polySia lectin EndoNDM, have been extensively used for studying polySia. However, EndoN is heat stable and remains associated with cells after washing. When studying polySia in systems with multiple polysialylated species, the residual EndoN that cannot be removed confounds data interpretation. We developed a strategy for site-specific immobilization of EndoN on streptavidin-coated magnetic beads. We showed that immobilizing EndoN allows for effective removal of the enzyme from samples, while retaining hydrolase activity. We used the same strategy to immobilize the polySia lectin EndoNDM, which enabled the enrichment of polysialylated proteins from complex mixtures such as serum for their identification via mass spectrometry. We used this methodology to identify a novel polysialylated protein, QSOX2, which is secreted from the breast cancer cell line MCF-7. This method of site-specific immobilization can be utilized for other enzymes and lectins to yield insight into glycobiology.

Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis.

Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometrybased glycoproteomics datasets from human serum were shared with participants and the relative team performance for N- and O-glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved 'high-coverage' and 'high-accuracy' glycoproteomics search solutions. This study concludes that diverse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics.

Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice.

Neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19. Here, we identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2. Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody-Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.

CRISPRi-Mediated Silencing of Burkholderia O-Linked Glycosylation Systems Enables the Depletion of Glycosylation Yet Results in Modest Proteome Impacts.

The process of O-linked protein glycosylation is highly conserved across the Burkholderia genus and mediated by the oligosaccharyltransferase PglL. While our understanding of Burkholderia glycoproteomes has increased in recent years, little is known about how Burkholderia species respond to modulations in glycosylation. Utilizing CRISPR interference (CRISPRi), we explored the impact of silencing of O-linked glycosylation across four species of Burkholderia; Burkholderia cenocepacia K56-2, Burkholderia diffusa MSMB375, Burkholderia multivorans ATCC17616, and Burkholderia thailandensis E264. Proteomic and glycoproteomic analyses revealed that while CRISPRi enabled inducible silencing of PglL, this did not abolish glycosylation, nor recapitulate phenotypes such as proteome changes or alterations in motility that are associated with glycosylation null strains, despite inhibition of glycosylation by nearly 90%. Importantly, this work also demonstrated that CRISPRi induction with high levels of rhamnose leads to extensive impacts on the Burkholderia proteomes, which without appropriate controls mask the impacts specifically driven by CRISPRi guides. Combined, this work revealed that while CRISPRi allows the modulation of O-linked glycosylation with reductions up to 90% at a phenotypic and proteome levels, Burkholderia appears to demonstrate a robust tolerance to fluctuations in glycosylation capacity.

Discovery and characterization of a new class of O-linking oligosaccharyltransferases from the Moraxellaceae family.

Bacterial protein glycosylation is commonly mediated by oligosaccharyltransferases (OTases) that transfer oligosaccharides en bloc from preassembled lipid-linked precursors to acceptor proteins. Natively, O-linking OTases usually transfer a single repeat unit of the O-antigen or capsular polysaccharide to the side chains of serine or threonine on acceptor proteins. Three major families of bacterial O-linking OTases have been described: PglL, PglS, and TfpO. TfpO is limited to transferring short oligosaccharides both in its native context and when heterologously expressed in glycoengineered Escherichia coli. On the other hand, PglL and PglS can transfer long-chain polysaccharides when expressed in glycoengineered E. coli. Herein, we describe the discovery and functional characterization of a novel family of bacterial O-linking OTases termed TfpM from Moraxellaceae bacteria. TfpM proteins are similar in size and sequence to TfpO enzymes but can transfer long-chain polysaccharides to acceptor proteins. Phylogenetic analyses demonstrate that TfpM proteins cluster in distinct clades from known bacterial OTases. Using a representative TfpM enzyme from Moraxella osloensis, we determined that TfpM glycosylates a C-terminal threonine of its cognate pilin-like protein and identified the minimal sequon required for glycosylation. We further demonstrated that TfpM has broad substrate tolerance and can transfer diverse glycans including those with glucose, galactose, or 2-N-acetyl sugars at the reducing end. Last, we find that a TfpM-derived bioconjugate is immunogenic and elicits serotype-specific polysaccharide IgG responses in mice. The glycan substrate promiscuity of TfpM and identification of the minimal TfpM sequon renders this enzyme a valuable additional tool for expanding the glycoengineering toolbox.

Widespread Family of NAD+-Dependent Sulfoquinovosidases at the Gateway to Sulfoquinovose Catabolism.

The sulfosugar sulfoquinovose (SQ) is produced by photosynthetic plants, algae, and cyanobacteria on a scale of 10 billion tons per annum. Its degradation, which is essential to allow cycling of its constituent carbon and sulfur, involves specialized glycosidases termed sulfoquinovosidases (SQases), which release SQ from sulfolipid glycoconjugates, so SQ can enter catabolism pathways. However, many SQ catabolic gene clusters lack a gene encoding a classical SQase. Here, we report the discovery of a new family of SQases that use an atypical oxidoreductive mechanism involving NAD+ as a catalytic cofactor. Three-dimensional X-ray structures of complexes with SQ and NAD+ provide insight into the catalytic mechanism, which involves transient oxidation at C3. Bioinformatic survey reveals this new family of NAD+-dependent SQases occurs within sulfoglycolytic and sulfolytic gene clusters that lack classical SQases and is distributed widely including within Roseobacter clade bacteria, suggesting an important contribution to marine sulfur cycling.