RESUMEN

Correct elaboration of N-linked glycans in the secretory pathway of human cells is essential in physiology. Early N-glycan biosynthesis follows an assembly line principle before undergoing crucial elaboration points that feature the sequential incorporation of the sugar N-acetylglucosamine (GlcNAc). The activity of GlcNAc transferase V (MGAT5) primes the biosynthesis of an N-glycan antenna that is heavily upregulated in cancer. Still, the functional relevance and substrate choice of MGAT5 are ill-defined. Here, we employ protein engineering to develop a bioorthogonal substrate analog for the activity of MGAT5. Chemoenzymatic synthesis is used to produce a collection of nucleotide-sugar analogs with bulky, bioorthogonal acylamide side chains. We find that WT-MGAT5 displays considerable activity toward such substrate analogues. Protein engineering yields an MGAT5 variant that loses activity against the native nucleotide sugar and increases activity toward a 4-azidobutyramide-containing substrate analogue. By such restriction of substrate specificity, we show that the orthogonal enzyme-substrate pair is suitable to bioorthogonally tag glycoproteins. Through X-ray crystallography and molecular dynamics simulations, we establish the structural basis of MGAT5 engineering, informing the design rules for bioorthogonal precision chemical tools.

RESUMEN

[This corrects the article DOI: 10.1039/D3SC01594G.].

RESUMEN

The modification of nucleocytoplasmic proteins by O-linked N-acetylglucosamine (O-GlcNAc) is an important regulator of cell physiology. O-GlcNAc is installed on over a thousand proteins by just one enzyme, O-GlcNAc transferase (OGT). How OGT is regulated is therefore a topic of interest. To gain insight into these questions, we used OGT to perform phage display selection from an unbiased library of ~109 peptides of 15 amino acids in length. Following rounds of selection and deep mutational panning, we identified a high-fidelity peptide consensus sequence, [Y/F]-x-P-x-Y-x-[I/M/F], that drives peptide binding to OGT. Peptides containing this sequence bind to OGT in the high nanomolar to low micromolar range and inhibit OGT in a noncompetitive manner with low micromolar potencies. X-ray structural analyses of OGT in complex with a peptide containing this motif surprisingly revealed binding to an exosite proximal to the active site of OGT. This structure defines the detailed molecular basis driving peptide binding and explains the need for specific residues within the sequence motif. Analysis of the human proteome revealed this motif within 52 nuclear and cytoplasmic proteins. Collectively, these data suggest a mode of regulation of OGT by which polypeptides can bind to this exosite to cause allosteric inhibition of OGT through steric occlusion of its active site. We expect that these insights will drive improved understanding of the regulation of OGT within cells and enable the development of new chemical tools to exert fine control over OGT activity.

Asunto(s)

RESUMEN

Sulfolactate (SL) is a short-chain organosulfonate that is an important reservoir of sulfur in the biosphere. SL is produced by oxidation of sulfolactaldehyde (SLA), which in turn derives from sulfoglycolysis of the sulfosugar sulfoquinovose, or through oxidation of 2,3-dihydroxypropanesulfonate. Oxidation of SLA is catalyzed by SLA dehydrogenases belonging to the aldehyde dehydrogenase superfamily. We report that SLA dehydrogenase RlGabD from the sulfoglycolytic bacterium Rhizobium leguminsarum SRDI565 can use both NAD+ and NADP+ as cofactor to oxidize SLA, and indicatively operates through a rapid equilibrium ordered mechanism. We report the cryo-EM structure of RlGabD bound to NADH, revealing a tetrameric quaternary structure and supporting proposal of organosulfonate binding residues in the active site, and a catalytic mechanism. Sequence based homology searches identified SLA dehydrogenase homologs in a range of putative sulfoglycolytic gene clusters in bacteria predominantly from the phyla Actinobacteria, Firmicutes, and Proteobacteria. This work provides a structural and biochemical view of SLA dehydrogenases to complement our knowledge of SLA reductases, and provide detailed insights into a critical step in the organosulfur cycle.

RESUMEN

Glycosyltransferases are a superfamily of enzymes that are notoriously difficult to inhibit. Here we apply an mRNA display technology integrated with genetic code reprogramming, referred to as the RaPID (random non-standard peptides integrated discovery) system, to identify macrocyclic peptides with high binding affinities for O-GlcNAc transferase (OGT). These macrocycles inhibit OGT activity through an allosteric mechanism that is driven by their binding to the tetratricopeptide repeats of OGT. Saturation mutagenesis in a maturation screen using 39 amino acids, including 22 non-canonical residues, led to an improved unnatural macrocycle that is ≈40 times more potent than the parent compound (Ki app =1.5 nM). Subsequent derivatization delivered a biotinylated derivative that enabled one-step affinity purification of OGT from complex samples. The high potency and novel mechanism of action of these OGT ligands should enable new approaches to elucidate the specificity and regulation of OGT.

Asunto(s)

RESUMEN

Primary familial brain calcification (PFBC) is characterised by abnormal deposits of calcium phosphate within various regions of the brain that are associated with severe cognitive impairments, psychiatric conditions, and movement disorders. Recent studies in diverse populations have shown a link between mutations in myogenesis-regulating glycosidase (MYORG) and the development of this disease. MYORG is a member of glycoside hydrolase (GH) family 31 (GH31) and, like the other mammalian GH31 enzyme α-glucosidase II, this enzyme is found in the lumen of the endoplasmic reticulum (ER). Though presumed to act as an α-glucosidase due to its localization and sequence relatedness to α-glucosidase II, MYORG has never been shown to exhibit catalytic activity. Here, we show that MYORG is an α-galactosidase and present the high-resolution crystal structure of MYORG in complex with substrate and inhibitor. Using these structures, we map detrimental mutations that are associated with MYORG-associated brain calcification and define how these mutations may drive disease progression through loss of enzymatic activity. Finally, we also detail the thermal stabilisation of MYORG afforded by a clinically approved small molecule ligand, opening the possibility of using pharmacological chaperones to enhance the activity of mutant forms of MYORG.

Asunto(s)

RESUMEN

Enzymatic hydrolysis of α-L-fucose from fucosylated glycoconjugates is consequential in bacterial infections and the neurodegenerative lysosomal storage disorder fucosidosis. Understanding human α-L-fucosidase catalysis, in an effort toward drug design, has been hindered by the absence of three-dimensional structural data for any animal fucosidase. Here, we have used cryoelectron microscopy (cryo-EM) to determine the structure of human lysosomal α-L-fucosidase (FucA1) in both an unliganded state and in complex with the inhibitor deoxyfuconojirimycin. These structures, determined at 2.49 Å resolution, reveal the homotetrameric structure of FucA1, the architecture of the catalytic center, and the location of both natural population variations and disease-causing mutations. Furthermore, this work has conclusively identified the hitherto contentious identity of the catalytic acid/base as aspartate-276, representing a shift from both the canonical glutamate acid/base residue and a previously proposed glutamate residue. These findings have furthered our understanding of how FucA1 functions in both health and disease.

Asunto(s)

RESUMEN

The O-linked ß-N-acetylglucosamine modification is a core signalling mechanism, with erroneous patterns leading to cancer and neurodegeneration. Although thousands of proteins are subject to this modification, only a single essential glycosyltransferase catalyses its installation, the O-GlcNAc transferase, OGT. Previous studies have provided truncated structures of OGT through X-ray crystallography, but the full-length protein has never been observed. Here, we report a 5.3 Å cryo-EM model of OGT. We show OGT is a dimer, providing a structural basis for how some X-linked intellectual disability mutations at the interface may contribute to disease. We observe that the catalytic section of OGT abuts a 13.5 tetratricopeptide repeat unit region and find the relative positioning of these sections deviate from the previously proposed, X-ray crystallography-based model. We also note that OGT exhibits considerable heterogeneity in tetratricopeptide repeat units N-terminal to the dimer interface with repercussions for how OGT binds protein ligands and partners.

Asunto(s)

RESUMEN

The asymmetric Gram-negative outer membrane (OM) is the first line of defense for bacteria against environmental insults and attack by antimicrobials. The key component of the OM is lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system component LptD is regulated by a periplasmic metalloprotease, BepA. Here, we present the crystal structure of BepA from Escherichia coli, solved to a resolution of 2.18 Å, in which the M48 protease active site is occluded by an active-site plug. Informed by our structure, we demonstrate that free movement of the active-site plug is essential for BepA function, suggesting that the protein is autoregulated by the active-site plug, which is conserved throughout the M48 metalloprotease family. Targeted mutagenesis of conserved residues reveals that the negative pocket and the tetratricopeptide repeat (TPR) cavity are required for function and degradation of the BAM complex component BamA under conditions of stress. Last, we show that loss of BepA causes disruption of OM lipid asymmetry, leading to surface exposed phospholipid.IMPORTANCE M48 metalloproteases are widely distributed in all domains of life. E. coli possesses four members of this family located in multiple cellular compartments. The functions of these proteases are not well understood. Recent investigations revealed that one family member, BepA, has an important role in the maturation of a central component of the lipopolysaccharide (LPS) biogenesis machinery. Here, we present the structure of BepA and the results of a structure-guided mutagenesis strategy, which reveal the key residues required for activity that inform how all M48 metalloproteases function.

Asunto(s)

RESUMEN

Antimicrobial-resistant (AMR) infections pose a serious risk to human and animal health. A major factor contributing to this global crisis is the sharing of resistance genes between different bacteria via plasmids. The WHO lists Enterobacteriaceae, such as Escherichia coli and Klebsiella pneumoniae, producing extended-spectrum ß-lactamases (ESBL) and carbapenemases as "critical" priorities for new drug development. These resistance genes are most often shared via plasmid transfer. However, finding methods to prevent resistance gene sharing has been hampered by the lack of screening systems for medium-/high-throughput approaches. Here, we have used an ESBL-producing plasmid, pCT, and a carbapenemase-producing plasmid, pKpQIL, in two different Gram-negative bacteria, E. coli and K. pneumoniae Using these critical resistance-pathogen combinations, we developed an assay using fluorescent proteins, flow cytometry, and confocal microscopy to assess plasmid transmission inhibition within bacterial populations in a medium-throughput manner. Three compounds with some reports of antiplasmid properties were tested; chlorpromazine reduced transmission of both plasmids and linoleic acid reduced transmission of pCT. We screened the Prestwick library of over 1,200 FDA-approved drugs/compounds. From this, we found two nucleoside analogue drugs used to treat HIV, abacavir and azidothymidine (AZT), which reduced plasmid transmission (AZT, e.g., at 0.25 µg/ml reduced pCT transmission in E. coli by 83.3% and pKpQIL transmission in K. pneumoniae by 80.8% compared to untreated controls). Plasmid transmission was reduced by concentrations of the drugs which are below peak serum concentrations and are achievable in the gastrointestinal tract. These drugs could be used to decolonize humans, animals, or the environment from AMR plasmids.IMPORTANCE More and more bacterial infections are becoming resistant to antibiotics. This has made treatment of many infections very difficult. One of the reasons this is such a large problem is that bacteria are able to share their genetic material with other bacteria, and these shared genes often include resistance to a variety of antibiotics, including some of our drugs of last resort. We are addressing this problem by using a fluorescence-based system to search for drugs that will stop bacteria from sharing resistance genes. We uncovered a new role for two drugs used to treat HIV and show that they are able to prevent the sharing of two different types of resistance genes in two unique bacterial strains. This work lays the foundation for future work to reduce the prevalence of resistant infections.

Asunto(s)

RESUMEN

Glycosyltransferases carry out important cellular functions in species ranging from bacteria to humans. Despite their essential roles in biology, simple and robust activity assays that can be easily applied to high-throughput screening for inhibitors of these enzymes have been challenging to develop. Herein, we report a bead-based strategy to measure the group-transfer activity of glycosyltransferases sensitively using simple fluorescence measurements, without the need for coupled enzymes or secondary reactions. We validate the performance and accuracy of the assay using O-GlcNAc transferase (OGT) as a model system through detailed Michaelis-Menten kinetic analysis of various substrates and inhibitors. Optimization of this assay and application to high-throughput screening enabled screening for inhibitors of OGT, leading to a novel inhibitory scaffold. We believe this assay will prove valuable not only for the study of OGT, but also more widely as a general approach for the screening of glycosyltransferases and other group-transfer enzymes.

Asunto(s)

RESUMEN

The bacterial second messenger cyclic-di-GMP is a widespread, prominent effector of lifestyle change. An example of this occurs in the predatory bacterium Bdellovibrio bacteriovorus, which cycles between free-living and intraperiplasmic phases after entering (and killing) another bacterium. The initiation of prey invasion is governed by DgcB (GGDEF enzyme) that produces cyclic-di-GMP in response to an unknown stimulus. Here, we report the structure of DgcB, and demonstrate that the GGDEF and sensory forkhead-associated (FHA) domains form an asymmetric dimer. Our structures indicate that the FHA domain is a consensus phosphopeptide sensor, and that the ligand for activation is surprisingly derived from the N-terminal region of DgcB itself. We confirm this hypothesis by determining the structure of a FHA:phosphopeptide complex, from which we design a constitutively-active mutant (confirmed via enzyme assays). Our results provide an understanding of the stimulus driving DgcB-mediated prey invasion and detail a unique mechanism of GGDEF enzyme regulation.

Asunto(s)

RESUMEN

Bacterial usage of the cyclic dinucleotide c-di-GMP is widespread, governing the transition between motile/sessile and unicellular/multicellular behaviors. There is limited information on c-di-GMP metabolism, particularly on regulatory mechanisms governing control of EAL c-di-GMP phosphodiesterases. Herein, we provide high-resolution structures for an EAL enzyme Bd1971, from the predatory bacterium Bdellovibrio bacteriovorus, which is controlled by a second signaling nucleotide, cAMP. The full-length cAMP-bound form reveals the sensory N-terminus to be a domain-swapped variant of the cNMP/CRP family, which in the cAMP-activated state holds the C-terminal EAL enzyme in a phosphodiesterase-active conformation. Using a truncation mutant, we trap both a half-occupied and inactive apo-form of the protein, demonstrating a series of conformational changes that alter juxtaposition of the sensory domains. We show that Bd1971 interacts with several GGDEF proteins (c-di-GMP producers), but mutants of Bd1971 do not share the discrete phenotypes of GGDEF mutants, instead having an elevated level of c-di-GMP, suggesting that the role of Bd1971 is to moderate these levels, allowing "action potentials" to be generated by each GGDEF protein to effect their specific functions.

Asunto(s)

RESUMEN

The global crisis of antibiotic resistance has reached a point where, if action is not taken, human medicine will enter a postantibiotic world and simple injuries could once again be life threatening. New antibiotics are needed urgently, but better use of existing agents is just as important. More appropriate use of antibiotics in medicine is vital, but the extensive use of antibiotics outside medical settings is often overlooked. Antibiotics are commonly used in animal husbandry, bee-keeping, fish farming and other forms of aquaculture, ethanol production, horticulture, antifouling paints, food preservation, and domestically. This provides multiple opportunities for the selection and spread of antibiotic-resistant bacteria. Given the current crisis, it is vital that the nonmedical use of antibiotics is critically examined and that any nonessential use halted.

Asunto(s)