RESUMEN
Linear, unbranched (1,3;1,4)-ß-glucans (mixed-linkage glucans or MLGs) are commonly found in the cell walls of grasses, but have also been detected in basal land plants, algae, fungi and bacteria. Here we show that two family GT2 glycosyltransferases from the Gram-positive bacterium Sarcina ventriculi are capable of synthesizing MLGs. Immunotransmission electron microscopy demonstrates that MLG is secreted as an exopolysaccharide, where it may play a role in organizing individual cells into packets that are characteristic of Sarcina species. Heterologous expression of these two genes shows that they are capable of producing MLGs in planta, including an MLG that is chemically identical to the MLG secreted from S. ventriculi cells but which has regularly spaced (1,3)-ß-linkages in a structure not reported previously for MLGs. The tandemly arranged, paralogous pair of genes are designated SvBmlgs1 and SvBmlgs2. The data indicate that MLG synthases have evolved different enzymic mechanisms for the incorporation of (1,3)-ß- and (1,4)-ß-glucosyl residues into a single polysaccharide chain. Amino acid variants associated with the evolutionary switch from (1,4)-ß-glucan (cellulose) to MLG synthesis have been identified in the active site regions of the enzymes. The presence of MLG synthesis in bacteria could prove valuable for large-scale production of MLG for medical, food and beverage applications.
Asunto(s)
Glicosiltransferasas , beta-Glucanos , Glicosiltransferasas/metabolismo , Glicosiltransferasas/genética , beta-Glucanos/metabolismo , Pared Celular/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Polisacáridos Bacterianos/biosíntesis , Polisacáridos Bacterianos/metabolismoRESUMEN
Phosphoethanolamine (pEtN) cellulose is a naturally occurring modified cellulose produced by several Enterobacteriaceae. The minimal components of the E. coli cellulose synthase complex include the catalytically active BcsA enzyme, an associated periplasmic semicircle of hexameric BcsB, as well as the outer membrane (OM)-integrated BcsC subunit containing periplasmic tetratricopeptide repeats (TPR). Additional subunits include BcsG, a membrane-anchored periplasmic pEtN transferase associated with BcsA, and BcsZ, a conserved periplasmic cellulase of unknown biological function. While events underlying the synthesis and translocation of cellulose by BcsA are well described, little is known about its pEtN modification and translocation across the cell envelope. We show that the N-terminal cytosolic domain of BcsA positions three copies of BcsG near the nascent cellulose polymer. Further, the terminal subunit of the BcsB semicircle tethers the N-terminus of a single BcsC protein to establish a trans-envelope secretion system. BcsC's TPR motifs bind a putative cello-oligosaccharide near the entrance to its OM pore. Additionally, we show that only the hydrolytic activity of BcsZ but not the subunit itself is necessary for cellulose secretion, suggesting a secretion mechanism based on enzymatic removal of mislocalized cellulose. Lastly, we introduce pEtN modification of cellulose in orthogonal cellulose biosynthetic systems by protein engineering.
RESUMEN
Capsular polysaccharides (CPSs) fortify the cell boundaries of many commensal and pathogenic bacteria1. Through the ABC-transporter-dependent biosynthesis pathway, CPSs are synthesized intracellularly on a lipid anchor and secreted across the cell envelope by the KpsMT ABC transporter associated with the KpsE and KpsD subunits1,2. Here we use structural and functional studies to uncover crucial steps of CPS secretion in Gram-negative bacteria. We show that KpsMT has broad substrate specificity and is sufficient for the translocation of CPSs across the inner bacterial membrane, and we determine the cell surface organization and localization of CPSs using super-resolution fluorescence microscopy. Cryo-electron microscopy analyses of the KpsMT-KpsE complex in six different states reveal a KpsE-encaged ABC transporter, rigid-body conformational rearrangements of KpsMT during ATP hydrolysis and recognition of a glycolipid inside a membrane-exposed electropositive canyon. In vivo CPS secretion assays underscore the functional importance of canyon-lining basic residues. Combined, our analyses suggest a molecular model of CPS secretion by ABC transporters.
Asunto(s)
Cápsulas Bacterianas , Proteínas de Escherichia coli , Escherichia coli , Polisacáridos Bacterianos , Adenosina Trifosfato/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/ultraestructura , Cápsulas Bacterianas/metabolismo , Cápsulas Bacterianas/química , Cápsulas Bacterianas/ultraestructura , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Microscopía por Crioelectrón , Escherichia coli/química , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestructura , Glucolípidos/química , Glucolípidos/metabolismo , Hidrólisis , Microscopía Fluorescente , Modelos Moleculares , Polisacáridos Bacterianos/metabolismo , Polisacáridos Bacterianos/química , Especificidad por SustratoRESUMEN
Plant cell walls contain a meshwork of cellulose fibers embedded into a matrix of other carbohydrate and non-carbohydrate-based biopolymers. This composite material exhibits extraordinary properties, from stretchable and pliable cell boundaries to solid protective shells. Cellulose, a linear glucose polymer, is synthesized and secreted across the plasma membrane by cellulose synthase (CesA). Plants express several CesA isoforms, with different subsets necessary for primary and secondary cell wall biogenesis. The produced cellulose chains can be organized into fibrillar structures and fibrillogenesis likely requires the supramolecular organization of CesAs into pseudo sixfold symmetric complexes (CSCs). Here, we structurally and functionally characterize a set of soybean (Gm) CesA isoforms implicated in primary cell wall biogenesis. Cryogenic electron microscopy analyses of catalytically active GmCesA1, GmCesA3, and GmCesA6 reveal their assembly into homotrimeric complexes, stabilized by a cytosolic plant conserved region. Contrasting secondary cell wall CesAs, a peripheral position of the C-terminal transmembrane helix creates a large, lipid-exposed lateral opening of the enzymes' cellulose-conducting transmembrane channels. Co-purification experiments reveal that homotrimers of different CesA isoforms interact in vitro and that this interaction is independent of the enzymes' N-terminal cytosolic domains. Our data suggest that cross-isoform interactions are mediated by the class-specific region, which forms a hook-shaped protrusion of the catalytic domain at the cytosolic water-lipid interface. Further, inter-isoform interactions lead to synergistic catalytic activity, suggesting increased cellulose biosynthesis upon homotrimer interaction. Combined, our structural and biochemical data favor a model by which homotrimers of different CesA isoforms assemble into a microfibril-producing CSC.
RESUMEN
Hyaluronan (HA), the essential [-3-GlcNAc-1-ß-4-GlcA-1-ß-]n matrix polysaccharide in vertebrates and molecular camouflage coating in select pathogens, is polymerized by "HA synthase" (HAS) enzymes. The first HAS identified three decades ago opened the window for new insights and biotechnological tools. This review discusses current understanding of HA biosynthesis, its biotechnological utility, and addresses some misconceptions in the literature. HASs are fascinating enzymes that polymerize two different UDP-activated sugars via different glycosidic linkages. Therefore, these catalysts were the first examples to break the "one enzyme/one sugar transferred" dogma. Three distinct types of these bifunctional glycosyltransferases (GTs) with disparate architectures and reaction modes are known. Based on biochemical and structural work, we present an updated classification system. Class I membrane-integrated HASs employ a processive chain elongation mechanism and secrete HA across the plasma membrane. This complex operation is accomplished by functionally integrating a cytosolic catalytic domain with a channel-forming transmembrane region. Class I enzymes, containing a single GT family-2 (GT-2) module that adds both monosaccharide units to the nascent chain, are further subdivided into two groups that construct the polymer with opposite molecular directionalities: Class I-R and I-NR elongate the HA polysaccharide at either the reducing or the non-reducing end, respectively. In contrast, Class II HASs are membrane-associated peripheral synthases with a non-processive, non-reducing end elongation mechanism using two independent GT-2 modules (one for each type of monosaccharide) and require a separate secretion system for HA export. We discuss recent mechanistic insights into HA biosynthesis that promise biotechnological benefits and exciting engineering approaches.
Asunto(s)
Glucuronosiltransferasa , Glicosiltransferasas , Animales , Hialuronano Sintasas/genética , Glicosiltransferasas/genética , Glucuronosiltransferasa/química , Glucuronosiltransferasa/metabolismo , Ácido Hialurónico/química , Polisacáridos , Azúcares de Uridina Difosfato , MonosacáridosRESUMEN
Recent breakthroughs in structural biology have provided valuable new insights into enzymes involved in plant cell wall metabolism. More specifically, the molecular mechanism of synthesis of (1,3;1,4)-ß-glucans, which are widespread in cell walls of commercially important cereals and grasses, has been the topic of debate and intense research activity for decades. However, an inability to purify these integral membrane enzymes or apply transgenic approaches without interpretative problems associated with pleiotropic effects has presented barriers to attempts to define their synthetic mechanisms. Following the demonstration that some members of the CslF sub-family of GT2 family enzymes mediate (1,3;1,4)-ß-glucan synthesis, the expression of the corresponding genes in a heterologous system that is free of background complications has now been achieved. Biochemical analyses of the (1,3;1,4)-ß-glucan synthesized in vitro, combined with 3-dimensional (3D) cryogenic-electron microscopy and AlphaFold protein structure predictions, have demonstrated how a single CslF6 enzyme, without exogenous primers, can incorporate both (1,3)- and (1,4)-ß-linkages into the nascent polysaccharide chain. Similarly, 3D structures of xyloglucan endo-transglycosylases and (1,3;1,4)-ß-glucan endo- and exohydrolases have allowed the mechanisms of (1,3;1,4)-ß-glucan modification and degradation to be defined. X-ray crystallography and multi-scale modeling of a broad specificity GH3 ß-glucan exohydrolase recently revealed a previously unknown and remarkable molecular mechanism with reactant trajectories through which a polysaccharide exohydrolase can act with a processive action pattern. The availability of high-quality protein 3D structural predictions should prove invaluable for defining structures, dynamics, and functions of other enzymes involved in plant cell wall metabolism in the immediate future.
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beta-Glucanos , beta-Glucanos/metabolismo , Hidrólisis , Poaceae/metabolismo , Polisacáridos/metabolismo , Pared Celular/metabolismoRESUMEN
Cellulose is an abundant cell wall component of land plants. It is synthesized from UDP-activated glucose molecules by cellulose synthase, a membrane-integrated processive glycosyltransferase. Cellulose synthase couples the elongation of the cellulose polymer with its translocation across the plasma membrane. Here, we present substrate- and product-bound cryogenic electron microscopy structures of the homotrimeric cellulose synthase isoform-8 (CesA8) from hybrid aspen (poplar). UDP-glucose binds to a conserved catalytic pocket adjacent to the entrance to a transmembrane channel. The substrate's glucosyl unit is coordinated by conserved residues of the glycosyltransferase domain and amphipathic interface helices. Site-directed mutagenesis of a conserved gating loop capping the active site reveals its critical function for catalytic activity. Molecular dynamics simulations reveal prolonged interactions of the gating loop with the substrate molecule, particularly across its central conserved region. These transient interactions likely facilitate the proper positioning of the substrate molecule for glycosyl transfer and cellulose translocation.
Asunto(s)
Celulosa , Glucosiltransferasas , Celulosa/química , Glucosiltransferasas/química , Glucosa , Uridina DifosfatoRESUMEN
Polysaccharides are essential biopolymers produced in all kingdoms of life. On the cell surface, they represent versatile architectural components, forming protective capsules and coats, cell walls, or adhesives. Extracellular polysaccharide (EPS) biosynthesis mechanisms differ based on the cellular localization of polymer assembly. Some polysaccharides are first synthesized in the cytosol and then extruded by ATP powered transporters [1]. In other cases, the polymers are assembled outside the cell [2], synthesized and secreted in a single step [3], or deposited on the cell surface via vesicular trafficking [4]. This review focuses on recent insights into the biosynthesis, secretion, and assembly of EPS in microbes, plants and vertebrates. We focus on comparing the sites of biosynthesis, secretion mechanisms, and higher-order EPS assemblies.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Polisacáridos , Animales , Polisacáridos/metabolismo , Membrana Celular/metabolismo , Transporte Biológico , Pared Celular/metabolismoRESUMEN
Cellulose is an abundant cell wall component of land plants. It is synthesized from UDP-activated glucose molecules by cellulose synthase, a membrane-integrated processive glycosyltransferase. Cellulose synthase couples the elongation of the cellulose polymer with its translocation across the plasma membrane. Here, we present substrate and product-bound cryogenic electron microscopy structures of the homotrimeric cellulose synthase isoform-8 (CesA8) from hybrid aspen (poplar). UDP-glucose binds to a conserved catalytic pocket adjacent to the entrance to a transmembrane channel. The substrate's glucosyl unit is coordinated by conserved residues of the glycosyltransferase domain and amphipathic interface helices. Site-directed mutagenesis of a conserved gating loop capping the active site reveals its critical function for catalytic activity. Molecular dynamics simulations reveal prolonged interactions of the gating loop with the substrate molecule, particularly across its central conserved region. These transient interactions likely facilitate the proper positioning of the substrate molecule for glycosyl transfer and cellulose translocation. Highlights: Cryo-EM structures of substrate and product bound poplar cellulose synthase provide insights into substrate selectivitySite directed mutagenesis signifies a critical function of the gating loop for catalysisMolecular dynamics simulations support persistent gating loop - substrate interactionsGating loop helps in positioning the substrate molecule to facilitate cellulose elongationConserved cellulose synthesis substrate binding mechanism across the kingdoms.
RESUMEN
Mixed-linkage (1,3;1,4)-ß-glucans, which are widely distributed in cell walls of the grasses, are linear glucose polymers containing predominantly (1,4)-ß-linked glucosyl units interspersed with single (1,3)-ß-linked glucosyl units. Their distribution in cereal grains and unique structures are important determinants of dietary fibers that are beneficial to human health. We demonstrate that the barley cellulose synthase-like CslF6 enzyme is sufficient to synthesize a high-molecular weight (1,3;1,4)-ß-glucan in vitro. Biochemical and cryo-electron microscopy analyses suggest that CslF6 functions as a monomer. A conserved "switch motif" at the entrance of the enzyme's transmembrane channel is critical to generate (1,3)-linkages. There, a single-point mutation markedly reduces (1,3)-linkage formation, resulting in the synthesis of cellulosic polysaccharides. Our results suggest that CslF6 monitors the orientation of the nascent polysaccharide's second or third glucosyl unit. Register-dependent interactions with these glucosyl residues reposition the polymer's terminal glucosyl unit to form either a (1,3)- or (1,4)-ß-linkage.
Asunto(s)
Hordeum , beta-Glucanos , Humanos , Hordeum/genética , Microscopía por Crioelectrón , GlucanosRESUMEN
Cellulose biosynthesis in sessile bacterial colonies originates in the membrane-integrated bacterial cellulose synthase (Bcs) AB complex. We utilize optical tweezers to measure single-strand cellulose biosynthesis by BcsAB from Rhodobacter sphaeroides. Synthesis depends on uridine diphosphate glucose, Mg2+, and cyclic diguanosine monophosphate, with the last displaying a retention time of â¼80 min. Below a stall force of 12.7 pN, biosynthesis is relatively insensitive to force and proceeds at a rate of one glucose addition every 2.5 s at room temperature, increasing to two additions per second at 37°. At low forces, conformational hopping is observed. Single-strand cellulose stretching unveiled a persistence length of 6.2 nm, an axial stiffness of 40.7 pN, and an ability for complexes to maintain a tight grip, with forces nearing 100 pN. Stretching experiments exhibited hysteresis, suggesting that cellulose microstructure underpinning robust biofilms begins to form during synthesis. Cellohexaose spontaneously binds to nascent single cellulose strands, impacting polymer mechanical properties and increasing BcsAB activity.
Asunto(s)
Rhodobacter sphaeroides , Uridina Difosfato Glucosa , Metabolismo de los Hidratos de Carbono , Celulosa/metabolismo , Glucosa/metabolismo , Rhodobacter sphaeroides/metabolismo , Uridina Difosfato Glucosa/metabolismoRESUMEN
O antigens are ubiquitous protective extensions of lipopolysaccharides in the extracellular leaflet of the Gram-negative outer membrane. Following biosynthesis in the cytosol, the lipid-linked polysaccharide is transported to the periplasm by the WzmWzt ABC transporter. Often, O antigen secretion requires the chemical modification of its elongating terminus, which the transporter recognizes via a carbohydrate-binding domain (CBD). Here, using components from A. aeolicus, we identify the O antigen structure with methylated mannose or rhamnose as its cap. Crystal and cryo electron microscopy structures reveal how WzmWzt recognizes this cap between its carbohydrate and nucleotide-binding domains in a nucleotide-free state. ATP binding induces drastic conformational changes of its CBD, terminating interactions with the O antigen. ATPase assays and site directed mutagenesis reveal reduced hydrolytic activity upon O antigen binding, likely to facilitate polymer loading into the ABC transporter. Our results elucidate critical steps in the recognition and translocation of polysaccharides by ABC transporters.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Antígenos O , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Hidrólisis , Antígenos O/químicaRESUMEN
Hyaluronan is an acidic heteropolysaccharide comprising alternating N-acetylglucosamine and glucuronic acid sugars that is ubiquitously expressed in the vertebrate extracellular matrix1. The high-molecular-mass polymer modulates essential physiological processes in health and disease, including cell differentiation, tissue homeostasis and angiogenesis2. Hyaluronan is synthesized by a membrane-embedded processive glycosyltransferase, hyaluronan synthase (HAS), which catalyses the synthesis and membrane translocation of hyaluronan from uridine diphosphate-activated precursors3,4. Here we describe five cryo-electron microscopy structures of a viral HAS homologue at different states during substrate binding and initiation of polymer synthesis. Combined with biochemical analyses and molecular dynamics simulations, our data reveal how HAS selects its substrates, hydrolyses the first substrate to prime the synthesis reaction, opens a hyaluronan-conducting transmembrane channel, ensures alternating substrate polymerization and coordinates hyaluronan inside its transmembrane pore. Our research suggests a detailed model for the formation of an acidic extracellular heteropolysaccharide and provides insights into the biosynthesis of one of the most abundant and essential glycosaminoglycans in the human body.
Asunto(s)
Hialuronano Sintasas , Ácido Hialurónico , Phycodnaviridae , Microscopía por Crioelectrón , Hialuronano Sintasas/metabolismo , Phycodnaviridae/enzimología , PolímerosRESUMEN
In land plants and algae, cellulose is important for strengthening cell walls and preventing breakage due to physical forces. Though our understanding of cellulose production by cellulose synthases (CESAs) has seen significant advances for several land plant and bacterial species, functional characterization of this fundamental protein is absent in red algae. Here we identify CESA gene candidates in the calcifying red alga Calliarthron tuberculosum using sequence similarity-based approaches, and elucidate their phylogenetic relationship with other CESAs from diverse taxa. One gene candidate, CtCESA1, was closely related to other putative red algal CESA genes. To test if CtCESA1 encoded a true cellulose synthase, CtCESA1 protein was expressed and purified from insect and yeast expression systems. CtCESA1 showed glucan synthase activity in glucose tracer assays. CtCESA1 activity was relatively low when compared with plant and bacterial CESA activity. In an in vitro assay, a predicted N-terminal starch-binding domain from CtCESA1 bound red algal floridean starch extracts, representing a unique domain in red algal CESAs not present in CESAs from other lineages. When the CtCESA1 gene was introduced into Arabidopsis thaliana cesa mutants, the red algal CtCESA1 partially rescued the growth defects of the primary cell wall cesa6 mutant, but not cesa3 or secondary cell wall cesa7 mutants. A fluorescently tagged CtCESA1 localized to the plasma membrane in the Arabidopsis cesa6 mutant background. This study presents functional evidence validating the sequence annotation of red algal CESAs. The relatively low activity of CtCESA1, partial complementation in Arabidopsis, and presence of unique protein domains suggest that there are probably functional differences between the algal and land plant CESAs.
Asunto(s)
Glucosiltransferasas , Rhodophyta , Pared Celular/metabolismo , Glucosiltransferasas/metabolismo , Filogenia , Rhodophyta/enzimología , Rhodophyta/genéticaRESUMEN
Cellulose is frequently found in communities of sessile bacteria called biofilms. Escherichia coli and other enterobacteriaceae modify cellulose with phosphoethanolamine (pEtN) to promote host tissue adhesion. The E. coli pEtN cellulose biosynthesis machinery contains the catalytic BcsA-B complex that synthesizes and secretes cellulose, in addition to five other subunits. The membrane-anchored periplasmic BcsG subunit catalyzes pEtN modification. Here we present the structure of the roughly 1 MDa E. coli Bcs complex, consisting of one BcsA enzyme associated with six copies of BcsB, determined by single-particle cryo-electron microscopy. BcsB homo-oligomerizes primarily through interactions between its carbohydrate-binding domains as well as intermolecular beta-sheet formation. The BcsB hexamer creates a half spiral whose open side accommodates two BcsG subunits, directly adjacent to BcsA's periplasmic channel exit. The cytosolic BcsE and BcsQ subunits associate with BcsA's regulatory PilZ domain. The macrocomplex is a fascinating example of cellulose synthase specification.
Asunto(s)
Celulosa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Biocatálisis , Microscopía por Crioelectrón , Proteínas de Escherichia coli/ultraestructura , Modelos Moleculares , Complejos Multienzimáticos/ultraestructura , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Reproducibilidad de los ResultadosRESUMEN
O antigens are important cell surface polysaccharides in gram-negative bacteria where they extend core lipopolysaccharides in the extracellular leaflet of the outer membrane. O antigen structures are serotype specific and form extended cell surface barriers endowing many pathogens with survival benefits. In the ABC transporter-dependent biosynthesis pathway, O antigens are assembled on the cytosolic side of the inner membrane on a lipid anchor and reoriented to the periplasmic leaflet by the channel-forming WzmWzt ABC transporter for ligation to the core lipopolysaccharides. In many cases, this process depends on the chemical modification of the O antigen's nonreducing terminus, sensed by WzmWzt via a carbohydrate-binding domain (CBD) that extends its nucleotide-binding domain (NBD). Here, we provide the cryo-electron microscopy structure of the full-length WzmWzt transporter from Aquifex aeolicus bound to adenosine triphosphate (ATP) and in a lipid environment, revealing a highly asymmetric transporter organization. The CBDs dimerize and associate with only one NBD. Conserved loops at the CBD dimer interface straddle a conserved peripheral NBD helix. The CBD dimer is oriented perpendicularly to the NBDs and its putative ligand-binding sites face the transporter to likely modulate ATPase activity upon O antigen binding. Further, our structure reveals a closed WzmWzt conformation in which an aromatic belt near the periplasmic channel exit seals the transporter in a resting, ATP-bound state. The sealed transmembrane channel is asymmetric, with one open and one closed cytosolic and periplasmic portal. The structure provides important insights into O antigen recruitment to and translocation by WzmWzt and related ABC transporters.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Antígenos O/biosíntesis , Adenosina Trifosfato/metabolismo , Sitios de Unión , Transporte Biológico , Membrana Celular/metabolismo , Microscopía por Crioelectrón/métodos , Hidrólisis , Lipopolisacáridos/metabolismo , Antígenos O/metabolismo , Periplasma/metabolismo , Dominios ProteicosRESUMEN
Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/clasificación , Dominios Proteicos , Transportadoras de Casetes de Unión a ATP/metabolismo , Pliegue de ProteínaRESUMEN
Cellulose is an essential plant cell wall component and represents the most abundant biopolymer on Earth. Supramolecular plant cellulose synthase complexes organize multiple linear glucose polymers into microfibrils as load-bearing wall components. We determined the structure of a poplar cellulose synthase CesA homotrimer that suggests a molecular basis for cellulose microfibril formation. This complex, stabilized by cytosolic plant-conserved regions and helical exchange within the transmembrane segments, forms three channels occupied by nascent cellulose polymers. Secretion steers the polymers toward a common exit point, which could facilitate protofibril formation. CesA's N-terminal domains assemble into a cytosolic stalk that interacts with a microtubule-tethering protein and may thus be involved in CesA localization. Our data suggest how cellulose synthase complexes assemble and provide the molecular basis for plant cell wall engineering.
Asunto(s)
Glucosiltransferasas/química , Complejos Multienzimáticos/química , Proteínas de Plantas/química , Populus/enzimología , Biocatálisis , Dominio Catalítico , Multimerización de ProteínaRESUMEN
Complex carbohydrates are essential for many biological processes, from protein quality control to cell recognition, energy storage, and cell wall formation. Many of these processes are performed in topologically extracellular compartments or on the cell surface; hence, diverse secretion systems evolved to transport the hydrophilic molecules to their sites of action. Polyprenyl lipids serve as ubiquitous anchors and facilitators of these transport processes. Here, we summarize and compare bacterial biosynthesis pathways relying on the recognition and transport of lipid-linked complex carbohydrates. In particular, we compare transporters implicated in O antigen and capsular polysaccharide biosyntheses with those facilitating teichoic acid and N-linked glycan transport. Further, we discuss recent insights into the generation, recognition, and recycling of polyprenyl lipids.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Glucolípidos/biosíntesis , Antígenos O/biosíntesis , Poliprenoles/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico , Ligasas de Carbono-Oxígeno/química , Ligasas de Carbono-Oxígeno/genética , Ligasas de Carbono-Oxígeno/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glicosiltransferasas/química , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Modelos Moleculares , Estructura Secundaria de Proteína , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Ácidos Teicoicos/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismoRESUMEN
A hallmark of Gram-negative bacteria is an asymmetric outer membrane containing lipopolysaccharides (LPSs) in the extracellular leaflet. LPS molecules consist of lipid A, which is connected to the inner and outer core oligosaccharides. This LPS core structure is extended in the periplasm by the O antigen, a variable and serotype-defining polysaccharide. In the ABC transporter-dependent LPS biosynthesis pathway, the WzmWzt transporter secretes the complete O antigen across the inner membrane for ligation to the LPS core. In some O antigen transporters, the nucleotide-binding domain of Wzt is fused C-terminally to a carbohydrate-binding domain (CBD) that interacts with the O antigen chain. Here, we present the crystal structure of the Aquifex aeolicus CBD that reveals a conserved flat and a variable twisted jelly-roll surface. The CBD dimer is stabilized by mutual ß strand exchange. Microbial glycan array binding studies with the isolated CBD provide insights into its interaction with complex carbohydrates.