ABSTRACT
ß-Mannans are plant cell wall polysaccharides that are commonly found in human diets. However, a mechanistic understanding into the key populations that degrade this glycan is absent, especially for the dominant Firmicutes phylum. Here, we show that the prominent butyrate-producing Firmicute Roseburia intestinalis expresses two loci conferring metabolism of ß-mannans. We combine multi-"omic" analyses and detailed biochemical studies to comprehensively characterize loci-encoded proteins that are involved in ß-mannan capturing, importation, de-branching and degradation into monosaccharides. In mixed cultures, R. intestinalis shares the available ß-mannan with Bacteroides ovatus, demonstrating that the apparatus allows coexistence in a competitive environment. In murine experiments, ß-mannan selectively promotes beneficial gut bacteria, exemplified by increased R. intestinalis, and reduction of mucus-degraders. Our findings highlight that R. intestinalis is a primary degrader of this dietary fiber and that this metabolic capacity could be exploited to selectively promote key members of the healthy microbiota using ß-mannan-based therapeutic interventions.
Subject(s)
Clostridiales/metabolism , Dietary Carbohydrates/metabolism , Mannans/metabolism , Animals , Bacteroides/genetics , Bacteroides/metabolism , Clostridiales/enzymology , Clostridiales/genetics , Diet , Gastrointestinal Microbiome , Humans , Male , MiceABSTRACT
The N-terminal domain (residues 28-165) from the glycoside hydrolase family 10 from Roseburia intestinalis (RiCBMx), has been isotopically labeled and recombinantly expressed in Escherichia coli. Here we report 1H, 13C and 15N NMR chemical shift assignments for this carbohydrate binding module (CBM).
Subject(s)
Endo-1,4-beta Xylanases/chemistry , Firmicutes/enzymology , Nuclear Magnetic Resonance, Biomolecular , Receptors, Cell Surface/chemistry , Carbon Isotopes , Nitrogen Isotopes , Protein Structure, Secondary , ProtonsABSTRACT
Interactions of exopolysaccharides and proteins are of great importance in food science, but complicated to analyze and quantify at the molecular level. A surface plasmon resonance procedure was established to characterize binding of seven structure-determined, branched hetero-exopolysaccharides (HePSs) of 0.14-4.9MDa from lactic acid bacteria to different milk proteins (ß-casein, κ-casein, native and heat-treated ß-lactoglobulin) at pH 4.0-5.0. Maximum binding capacity (RUmax) and apparent affinity (KA,app) were HePS- and protein-dependent and varied for example 10- and 600-fold, respectively, in the complexation with native ß-lactoglobulin at pH 4.0. Highest RUmax and KA,app were obtained with heat-treated ß-lactoglobulin and ß-casein, respectively. Overall, RUmax and KA,app decreased 6- and 20-fold, respectively, with increasing pH from 4.0 to 5.0. KA,app was influenced by ionic strength and temperature, indicating that polar interactions stabilize HePS-protein complexes. HePS size as well as oligosaccharide repeat structure, conferring chain flexibility and hydrogen bonding potential, influence the KA,app.
Subject(s)
Lactobacillales , Milk Proteins/chemistry , Polysaccharides, Bacterial/chemistry , Caseins/chemistry , Hot Temperature , Hydrogen-Ion Concentration , Lactoglobulins/chemistry , Molecular WeightABSTRACT
The present study describes the growth of the very well-known probiotic bacterium Lactobacillus acidophilus NCFM on different carbohydrates. Furthermore, recombinant production of putative moonlighting proteins elongation factor G and pyruvate kinase from this bacterium is described. For further and detailed interpretation of the data presented here, please see the research article "Mucin- and carbohydrate-stimulated adhesion and subproteome changes of the probiotic bacterium Lactobacillus acidophilus NCFM" (Celebioglu et al., 2017) [1].
ABSTRACT
Lytic polysaccharide monooxygenases (LPMOs) are important for the enzymatic conversion of biomass and seem to play a key role in degradation of the plant cell wall. In this study, we characterize an LPMO from the fungal plant pathogen Fusarium graminearum (FgLPMO9A) that catalyzes the mixed C1/C4 oxidative cleavage of cellulose and xyloglucan, but is inactive toward other (1,4)-linked ß-glucans. Our findings indicate that FgLPMO9A has unprecedented broad specificity on xyloglucan, cleaving any glycosidic bond in the ß-glucan main chain, regardless of xylosyl substitutions. Interestingly, we found that when incubated with a mixture of xyloglucan and cellulose, FgLPMO9A efficiently attacks the xyloglucan, whereas cellulose conversion is inhibited. This suggests that removal of hemicellulose may be the true function of this LPMO during biomass conversion.
Subject(s)
Fungal Proteins/metabolism , Fusarium/enzymology , Glucans/metabolism , Mixed Function Oxygenases/metabolism , Xylans/metabolism , Cellulose/metabolism , Fungal Proteins/chemistry , Mixed Function Oxygenases/chemistry , Substrate SpecificityABSTRACT
An α-L-arabinofuranosidase of GH62 from Aspergillus nidulans FGSC A4 (AnAbf62A-m2,3) has an unusually high activity towards wheat arabinoxylan (WAX) (67 U/mg; k cat = 178/s, K m = 4.90 mg/ml) and arabinoxylooligosaccharides (AXOS) with degrees of polymerisation (DP) 3-5 (37-80 U/mg), but about 50 times lower activity for sugar beet arabinan and 4-nitrophenyl-α-L-arabinofuranoside. α-1,2- and α-1,3-linked arabinofuranoses are released from monosubstituted, but not from disubstituted, xylose in WAX and different AXOS as demonstrated by NMR and polysaccharide analysis by carbohydrate gel electrophoresis (PACE). Mutants of the predicted general acid (Glu(188)) and base (Asp(28)) catalysts, and the general acid pK a modulator (Asp(136)) lost 1700-, 165- and 130-fold activities for WAX. WAX, oat spelt xylan, birchwood xylan and barley ß-glucan retarded migration of AnAbf62A-m2,3 in affinity electrophoresis (AE) although the latter two are neither substrates nor inhibitors. Trp(23) and Tyr(44), situated about 30 Å from the catalytic site as seen in an AnAbf62A-m2,3 homology model generated using Streptomyces thermoviolaceus SthAbf62A as template, participate in carbohydrate binding. Compared to wild-type, W23A and W23A/Y44A mutants are less retarded in AE, maintain about 70 % activity towards WAX with K i of WAX substrate inhibition increasing 4-7-folds, but lost 77-96 % activity for the AXOS. The Y44A single mutant had less effect, suggesting Trp(23) is a key determinant. AnAbf62A-m2,3 seems to apply different polysaccharide-dependent binding modes, and Trp(23) and Tyr(44) belong to a putative surface binding site which is situated at a distance of the active site and has to be occupied to achieve full activity.
Subject(s)
Aspergillus nidulans/enzymology , Fungal Proteins/chemistry , Glycoside Hydrolases/chemistry , Xylans/chemistry , Arabinose/analogs & derivatives , Arabinose/chemistry , Aspergillus nidulans/genetics , Binding Sites , Catalytic Domain , Cloning, Molecular , Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Phylogeny , Pichia/genetics , Pichia/metabolism , Polysaccharides/chemistry , Protein Conformation , Streptomyces/genetics , Streptomyces/metabolism , Substrate Specificity , Triticum/chemistry , Xylose/chemistry , beta-Glucans/chemistryABSTRACT
Molecular details underlying regulation of starch mobilization in cereal seed endosperm remain unknown despite the paramount role of this process in plant growth. The structure of the complex between the starch debranching enzyme barley limit dextrinase (LD), hydrolyzing α-1,6-glucosidic linkages, and its endogenous inhibitor (LDI) was solved at 2.7 Å. The structure reveals an entirely new and unexpected binding mode of LDI as compared with previously solved complex structures of related cereal type family inhibitors (CTIs) bound to glycoside hydrolases but is structurally analogous to binding of dual specificity CTIs to proteases. Site-directed mutagenesis establishes that a hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to LD as assessed by analysis of binding by using surface plasmon resonance and also supported by LDI inhibition of the enzyme activity. A phylogenetic analysis identified four LDI-like proteins in cereals among the 45 sequences from monocot databases that could be classified as unique CTI sequences. The unprecedented binding mechanism shown here for LDI has likely evolved in cereals from a need for effective inhibition of debranching enzymes having characteristic open active site architecture. The findings give a mechanistic rationale for the potency of LD activity regulation and provide a molecular understanding of the debranching events associated with optimal starch mobilization and utilization during germination. This study unveils a hitherto not recognized structural basis for the features endowing diversity to CTIs.
Subject(s)
Enzyme Inhibitors/chemistry , Glycoside Hydrolases/chemistry , Hordeum/enzymology , Plant Proteins/chemistry , Catalytic Domain , Crystallography, X-Ray , Enzyme Inhibitors/metabolism , Germination/physiology , Glycoside Hydrolases/antagonists & inhibitors , Glycoside Hydrolases/genetics , Glycoside Hydrolases/metabolism , Hordeum/genetics , Mutagenesis, Site-Directed , Plant Proteins/antagonists & inhibitors , Plant Proteins/genetics , Plant Proteins/metabolism , Starch/chemistry , Starch/genetics , Starch/metabolism , Structure-Activity RelationshipABSTRACT
In this study, a simple purification protocol is developed to reduce the bovine serum albumin (BSA) content in commercially available bovine submaxillary mucin (BSM). This involved purification of the BSM by one-column anion-exchange chromatography protocol resulting in BSM with greatly reduced BSA content and homogeneously distributed size, and in a high yield of â¼43% from BSM as received from the manufacturer. The purity and composition of commercially acquired BSM were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry, which verified that BSA is the most abundant nonmucinous protein component. The purification effect was evident from a significantly altered circular dichroism (CD) spectrum of BSM after anion-exchange chromatography.
Subject(s)
Chromatography, Ion Exchange/methods , Mucins/isolation & purification , Animals , Cattle , Chromatography, Thin Layer , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Mucins/chemistry , Serum Albumin, Bovine/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
The present study aimed at examining oligosaccharides (OS) for potential stimulation of probiotic bacteria. Nineteen structurally well-defined candidate OS covering groups of ß-glucosides, α-glucosides and α-galactosides with degree of polymerization 2-4 were prepared in >100 mg amounts by chemoenzymatic synthesis (i.e. reverse phosphorolysis or transglycosylation). Fourteen of the OS are not naturally occurring and five (ß-D-glucosyl-fructose, ß-D-glucosyl-xylitol, α-glucosyl-(1,4)-D-mannose, α-glucosyl-(1,4)-D-xylose; α-glucosyl-(1,4)-L-fucose) have recently been synthesized for the first time. These OS have not been previously tested for effects of bacterial growth and here the ability of all 19 OS to support growth of four gastrointestinal bacteria: three probiotic bacteria Bifidobacterium lactis, Bifidobacterium longum, and Lactobacillus acidophilus, and one commensal bacterium, Bacteroides vulgatus has been evaluated in monocultures. The disaccharides ß-D-glucosyl-xylitol and ß-D-glucosyl-(1,4)-xylose noticeably stimulated growth yields of L. acidophilus NCFM, and additionally, ß-D-glucosyl-(1,4)-xylose stimulated B. longum Bl-05. α-Glucosyl-(1,4)-glucosamine and α-glucosyl-(1,4)-N-acetyl-glucosamine enhanced the growth rate of B. animalis subsp. lactis and B. longum Bl-05, whereas L. acidophilus NCFM and Bac. vulgatus did not grow on these OS. α-Galactosyl-(1,6)-α-galactosyl-(1,6)-glucose advanced the growth rate of B. animalis subsp. lactis and L. acidophilus NCFM. Thus several of the structurally well-defined OS supported growth of beneficial gut bacteria. This reflects a broad specificity of their sugar transporters for OS, including specificity for non-naturally occurring OS, hence showing promise for design of novel prebiotics.
Subject(s)
Bacteroides/growth & development , Bifidobacterium/growth & development , Gastrointestinal Tract/microbiology , Lactobacillus acidophilus/growth & development , Oligosaccharides/chemistry , Bacteroides/isolation & purification , Bifidobacterium/isolation & purification , Disaccharides/metabolism , Humans , Lactobacillus acidophilus/isolation & purification , Mannose/metabolism , Prebiotics/analysis , Probiotics , Xylose/metabolismABSTRACT
The human gastrointestinal tract can be positively modulated by dietary supplementation of probiotic bacteria in combination with prebiotic carbohydrates. Here differential transcriptomics and functional genomics were used to identify genes in Lactobacillus acidophilus NCFM involved in the uptake and catabolism of 11 potential prebiotic compounds consisting of α- and ß-linked galactosides and glucosides. These oligosaccharides induced genes encoding phosphoenolpyruvate-dependent sugar phosphotransferase systems (PTS), galactoside pentose hexuronide (GPH) permease, and ATP-binding cassette (ABC) transporters. PTS systems were upregulated primarily by di- and tri-saccharides such as cellobiose, isomaltose, isomaltulose, panose and gentiobiose, while ABC transporters were upregulated by raffinose, Polydextrose, and stachyose. A single GPH transporter was induced by lactitol and galactooligosaccharides (GOS). The various transporters were associated with a number of glycoside hydrolases from families 1, 2, 4, 13, 32, 36, 42, and 65, involved in the catabolism of various α- and ß-linked glucosides and galactosides. Further subfamily specialization was also observed for different PTS-associated GH1 6-phospho-ß-glucosidases implicated in the catabolism of gentiobiose and cellobiose. These findings highlight the broad oligosaccharide metabolic repertoire of L. acidophilus NCFM and establish a platform for selection and screening of both probiotic bacteria and prebiotic compounds that may positively influence the gastrointestinal microbiota.
Subject(s)
Gene Expression Regulation, Bacterial , Lactobacillus acidophilus/metabolism , Prebiotics , ATP-Binding Cassette Transporters/metabolism , Cellobiose/pharmacology , Gene Expression Regulation, Bacterial/drug effects , Glucans/pharmacology , Isomaltose/analogs & derivatives , Isomaltose/pharmacology , Lactobacillus acidophilus/drug effects , Lactobacillus acidophilus/enzymology , Oligosaccharides/pharmacology , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Raffinose/pharmacology , Sugar Alcohols/pharmacologyABSTRACT
Lactobacillus acidophilus NCFM is a probiotic bacterium known for its beneficial effects on human health. The importance of α-galactosidases (α-Gals) for growth of probiotic organisms on oligosaccharides of the raffinose family present in many foods is increasingly recognized. Here, the crystal structure of α-Gal from L. acidophilus NCFM (LaMel36A) of glycoside hydrolase (GH) family 36 (GH36) is determined by single-wavelength anomalous dispersion. In addition, a 1.58-Å-resolution crystallographic complex with α-d-galactose at substrate binding subsite -1 was determined. LaMel36A has a large N-terminal twisted ß-sandwich domain, connected by a long α-helix to the catalytic (ß/α)(8)-barrel domain, and a C-terminal ß-sheet domain. Four identical monomers form a tightly packed tetramer where three monomers contribute to the structural integrity of the active site in each monomer. Structural comparison of LaMel36A with the monomeric Thermotoga maritima α-Gal (TmGal36A) reveals that O2 of α-d-galactose in LaMel36A interacts with a backbone nitrogen in a glycine-rich loop of the catalytic domain, whereas the corresponding atom in TmGal36A is from a tryptophan side chain belonging to the N-terminal domain. Thus, two distinctly different structural motifs participate in substrate recognition. The tetrameric LaMel36A furthermore has a much deeper active site than the monomeric TmGal36A, which possibly modulates substrate specificity. Sequence analysis of GH36, inspired by the observed structural differences, results in four distinct subgroups having clearly different active-site sequence motifs. This novel subdivision incorporates functional and architectural features and may aid further biochemical and structural analyses within GH36.
Subject(s)
Galactose/chemistry , Galactose/metabolism , Lactobacillus acidophilus/enzymology , Protein Multimerization , alpha-Galactosidase/chemistry , alpha-Galactosidase/metabolism , Catalytic Domain , Crystallography, X-Ray , Lactobacillus acidophilus/chemistry , Models, Molecular , Phylogeny , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Thermotoga maritima/chemistry , Thermotoga maritima/enzymologyABSTRACT
The limit dextrinase inhibitor (LDI) from barley seeds acts specifically on limit dextrinase (LD), an endogenous starch debranching enzyme. LDI is a 14 kDa hydrophobic protein containing four disulfide bonds and one unpaired thiol group previously found to be either glutathionylated or cysteinylated. It is a member of the so-called CM-protein family that includes α-amylase and serine protease inhibitors, which have been extremely challenging to produce recombinantly in functional form and in good yields. Here, LDI is produced in very high yields by secretory expression by Pichia pastoris applying high cell-density fermentation in a 5L fed-batch bioreactor. Thus about 200mg of LDI, which showed twofold higher inhibitory activity towards LD than LDI from barley seeds, was purified from 1L of culture supernatant by His-tag affinity chromatography and gel filtration. Electrospray ionization mass spectrometry verified the identity of the produced glutathionylated LDI-His(6). At a 1:1M ratio the recombinant LDI completely inhibited hydrolysis of pullulan catalyzed by 5-10 nM LD. LDI retained stability in the pH 2-12 range and at pH 6.5 displayed a half-life of 53 and 33 min at 90 and 93°C, respectively. The efficient heterologous production of LDI suggests secretory expression by P. pastoris to be a promising strategy to obtain other recombinant CM-proteins.
Subject(s)
Enzyme Inhibitors/pharmacology , Glucans/antagonists & inhibitors , Glycoside Hydrolases/antagonists & inhibitors , Hordeum/enzymology , Plant Proteins/isolation & purification , Recombinant Proteins/isolation & purification , Seeds/enzymology , Bioreactors , Cell Count , Chromatography, Affinity , Chromatography, Gel , Cloning, Molecular , Enzyme Inhibitors/metabolism , Enzyme Stability , Fermentation , Glucans/metabolism , Glycoside Hydrolases/metabolism , Half-Life , Hot Temperature , Hydrogen-Ion Concentration , Hydrolysis/drug effects , Kinetics , Pichia , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Proteins/pharmacology , Plasmids , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Spectrometry, Mass, Electrospray Ionization , Transformation, GeneticABSTRACT
Two ß-xylosidases of glycoside hydrolase family 3 (GH 3) from Aspergillus nidulans FGSC A4, BxlA and BxlB were produced recombinantly in Pichia pastoris and secreted to the culture supernatants in yields of 16 and 118 mg/L, respectively. BxlA showed about sixfold higher catalytic efficiency (k(cat)/K(m)) than BxlB towards para-nitrophenyl ß-D-xylopyranoside (pNPX) and ß-1,4-xylo-oligosaccharides (degree of polymerisation 2-6). For both enzymes k(cat)/K(m) decreased with increasing ß-1,4-xylo-oligosaccharide chain length. Using pNPX as donor with 9 monosaccharides, 7 disaccharides and two sugar alcohols as acceptors 18 different ß-xylosyl-oligosaccharides were synthesised in 2-36% (BxlA) and 6-66% (BxlB) yields by transxylosylation. BxlA utilised the monosaccharides D-mannose, D-lyxose, D-talose, D-xylose, D-arabinose, L-fucose, D-glucose, D-galactose and D-fructose as acceptors, whereas BxlB used the same except for D-lyxose, D-arabinose and L-fucose. BxlB transxylosylated the disaccharides xylobiose, lactulose, sucrose, lactose and turanose in upto 35% yield, while BxlA gave inferior yields on these acceptors. The regioselectivity was acceptor dependent and primarily involved ß-1,4 or 1,6 product linkage formation although minor products with different linkages were also obtained. Five of the 18 transxylosylation products obtained from D-lyxose, D-galactose, turanose and sucrose (two products) as acceptors were novel xylosyl-oligosaccharides, ß-D-Xylp-(1â4)-D-Lyxp, ß-D-Xylp-(1â6)-D-Galp, ß-D-Xylp-(1â4)-α-D-Glcp-(1â3)-ß-D-Fruf, ß-D-Xylp-(1â4)-α-D-Glcp-(1â2)-ß-D-Fruf, and ß-D-Xylp-(1â6)-ß-D-Fruf-(2â1)-α-D-Glcp, as structure-determined by 2D NMR, indicating that GH3 ß-xylosidases are able to transxylosylate a larger variety of carbohydrate acceptors than earlier reported. Furthermore, transxylosylation of certain acceptors resulted in mixtures. Some of these products are also novel, but the structures of the individual products could not be determined.
Subject(s)
Aspergillus nidulans , Oligosaccharides/chemical synthesis , Recombinant Proteins/chemistry , Xylose/analogs & derivatives , Xylose/chemical synthesis , Xylosidases/chemistry , Catalysis , Disaccharides/chemical synthesis , Molecular Conformation , Molecular Structure , Molecular Weight , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Stereoisomerism , Trisaccharides/chemical synthesis , Xylosidases/biosynthesis , Xylosidases/isolation & purificationABSTRACT
Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from α-d-glucose 1-phosphate as donor with glucose and cellobiose as acceptor, respectively. Use of α-d-glucosyl 1-fluoride as donor increased product yields to 98% for CtCBP and 68% for CtCDP. CtCBP showed broad acceptor specificity forming ß-glucosyl disaccharides with ß-(1â4)- regioselectivity from five monosaccharides as well as branched ß-glucosyl trisaccharides with ß-(1â4)-regioselectivity from three (1â6)-linked disaccharides. CtCDP showed strict ß-(1â4)-regioselectivity and catalysed linear chain extension of the three ß-linked glucosyl disaccharides, cellobiose, sophorose, and laminaribiose, whereas 12 tested monosaccharides were not acceptors. Structure analysis by NMR and ESI-MS confirmed two ß-glucosyl oligosaccharide product series to represent novel compounds, i.e. ß-D-glucopyranosyl-[(1â4)-ß-D-glucopyranosyl](n)-(1â2)-D-glucopyranose, and ß-D-glucopyranosyl-[(1â4)-ß-D-glucopyranosyl](n)-(1â3)-D-glucopyranose (n = 1-7). Multiple sequence alignment together with a modelled CtCBP structure, obtained using the crystal structure of Cellvibrio gilvus CBP in complex with glucose as a template, indicated differences in the subsite +1 region that elicit the distinct acceptor specificities of CtCBP and CtCDP. Thus Glu636 of CtCBP recognized the C1 hydroxyl of ß-glucose at subsite +1, while in CtCDP the presence of Ala800 conferred more space, which allowed accommodation of C1 substituted disaccharide acceptors at the corresponding subsites +1 and +2. Furthermore, CtCBP has a short Glu496-Thr500 loop that permitted the C6 hydroxyl of glucose at subsite +1 to be exposed to solvent, whereas the corresponding longer loop Thr637-Lys648 in CtCDP blocks binding of C6-linked disaccharides as acceptors at subsite +1. High yields in chemoenzymatic synthesis, a novel regioselectivity, and novel oligosaccharides including products of CtCDP catalysed oligosaccharide oligomerisation using α-d-glucosyl 1-fluoride, all together contribute to the formation of an excellent basis for rational engineering of CBP and CDP to produce desired oligosaccharides.
Subject(s)
Bacterial Proteins/metabolism , Clostridium thermocellum/enzymology , Glucosyltransferases/metabolism , Oligosaccharides/biosynthesis , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites/genetics , Biocatalysis , Cellobiose/biosynthesis , Cellobiose/chemistry , Cellulose/analogs & derivatives , Cellulose/biosynthesis , Cellulose/chemistry , Chromatography, High Pressure Liquid , Clostridium thermocellum/genetics , Clostridium thermocellum/metabolism , Dextrins/biosynthesis , Dextrins/chemistry , Enzyme Stability , Glucosyltransferases/chemistry , Glucosyltransferases/genetics , Hydrogen-Ion Concentration , Models, Molecular , Molecular Sequence Data , Molecular Structure , Oligosaccharides/chemistry , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Stereoisomerism , TemperatureABSTRACT
The alpha-galactosidase from Aspergillus nidulans (AglC) belongs to a phylogenetic cluster containing eukaryotic alpha-galactosidases and alpha-galacto-oligosaccharide synthases of glycoside hydrolase family 36 (GH36). The recombinant AglC, produced in high yield (0.65 g.L(-1) culture) as His-tag fusion in Escherichia coli, catalysed efficient transglycosylation with alpha-(1-->6) regioselectivity from 40 mm 4-nitrophenol alpha-d-galactopyranoside, melibiose or raffinose, resulting in a 37-74% yield of 4-nitrophenol alpha-D-Galp-(1-->6)-D-Galp, alpha-D-Galp-(1-->6)-alpha-D-Galp-(1-->6)-D-Glcp and alpha-D-Galp-(1-->6)-alpha-D-Galp-(1-->6)-D-Glcp-(alpha1-->beta2)-d-Fruf (stachyose), respectively. Furthermore, among 10 monosaccharide acceptor candidates (400 mm) and the donor 4-nitrophenol alpha-D-galactopyranoside (40 mm), alpha-(1-->6) linked galactodisaccharides were also obtained with galactose, glucose and mannose in high yields of 39-58%. AglC did not transglycosylate monosaccharides without the 6-hydroxymethyl group, i.e. xylose, L-arabinose, L-fucose and L-rhamnose, or with axial 3-OH, i.e. gulose, allose, altrose and L-rhamnose. Structural modelling using Thermotoga maritima GH36 alpha-galactosidase as the template and superimposition of melibiose from the complex with human GH27 alpha-galactosidase supported that recognition at subsite +1 in AglC presumably requires a hydrogen bond between 3-OH and Trp358 and a hydrophobic environment around the C-6 hydroxymethyl group. In addition, successful transglycosylation of eight of 10 disaccharides (400 mm), except xylobiose and arabinobiose, indicated broad specificity for interaction with the +2 subsite. AglC thus transferred alpha-galactosyl to 6-OH of the terminal residue in the alpha-linked melibiose, maltose, trehalose, sucrose and turanose in 6-46% yield and the beta-linked lactose, lactulose and cellobiose in 28-38% yield. The product structures were identified using NMR and ESI-MS and five of the 13 identified products were novel, i.e. alpha-D-Galp-(1-->6)-D-Manp; alpha-D-Galp-(1-->6)-beta-D-Glcp-(1-->4)-D-Glcp; alpha-D-Galp-(1-->6)-beta-D-Galp-(1-->4)-D-Fruf; alpha-D-Galp-(1-->6)-D-Glcp-(alpha1-->alpha1)-D-Glcp; and alpha-D-Galp-(1-->6)-alpha-D-Glcp-(1-->3)-D-Fruf.
Subject(s)
Aspergillus nidulans/enzymology , Biocatalysis , Oligosaccharides/biosynthesis , alpha-Galactosidase/metabolism , Amino Acid Sequence , Carbohydrate Conformation , Cloning, Molecular , Escherichia coli/metabolism , Glycosylation , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Models, Molecular , Oligosaccharides/chemistry , Phylogeny , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Temperature , alpha-Galactosidase/biosynthesis , alpha-Galactosidase/isolation & purificationABSTRACT
Heterologous production of large multidomain proteins from higher plants is often cumbersome. Barley limit dextrinase (LD), a 98kDa multidomain starch and alpha-limit dextrin debranching enzyme, plays a major role in starch mobilization during seed germination and is possibly involved in starch biosynthesis by trimming of intermediate branched alpha-glucan structures. Highly active barley LD is obtained by secretory expression during high cell-density fermentation of Pichia pastoris. The LD encoding gene fragment without signal peptide was subcloned in-frame with the Saccharomyces cerevisiae alpha-factor secretion signal of the P. pastoris vector pPIC9K under control of the alcohol oxidase 1 promoter. Optimization of a fed-batch fermentation procedure enabled efficient production of LD in a 5-L bioreactor, which combined with affinity chromatography on beta-cyclodextrin-Sepharose followed by Hiload Superdex 200 gel filtration yielded 34 mg homogenous LD (84% recovery). The identity of the recombinant LD was verified by N-terminal sequencing and by mass spectrometric peptide mapping. A molecular mass of 98kDa was estimated by SDS-PAGE in excellent agreement with the theoretical value of 97419Da. Kinetic constants of LD catalyzed pullulan hydrolysis were found to K(m,app)=0.16+/-0.02 mg/mL and k(cat,app)=79+/-10s(-1) by fitting the uncompetitive substrate inhibition Michaelis-Menten equation, which reflects significant substrate inhibition and/or transglycosylation. The resulting catalytic coefficient, k(cat,app)/K(m,app)=488+/-23mL/(mgs) is 3.5-fold higher than for barley malt LD. Surface plasmon resonance analysis showed alpha-, beta-, and gamma-cyclodextrin binding to LD with K(d) of 27.2, 0.70, and 34.7 microM, respectively.
Subject(s)
Biotechnology/methods , Fermentation , Glycoside Hydrolases/metabolism , Hordeum/enzymology , Pichia/metabolism , Amino Acid Sequence , Base Sequence , Bioreactors , Cloning, Molecular , Cyclodextrins/metabolism , Electrophoresis, Polyacrylamide Gel , Glucans/metabolism , Glycoside Hydrolases/isolation & purification , Least-Squares Analysis , Molecular Sequence Data , Plasmids/chemistry , Plasmids/genetics , Recombinant Proteins/isolation & purification , Subcellular Fractions/metabolism , Surface Plasmon Resonance , Transformation, GeneticABSTRACT
The family 20 carbohydrate-binding module (CBM20) of the Arabidopsis starch phosphorylator glucan, water dikinase 3 (GWD3) was heterologously produced and its properties were compared to the CBM20 from a fungal glucoamylase (GA). The GWD3 CBM20 has 50-fold lower affinity for cyclodextrins than that from GA. Homology modelling identified possible structural elements responsible for this weak binding of the intracellular CBM20. Differential binding of fluorescein-labelled GWD3 and GA modules to starch granules in vitro was demonstrated by confocal laser scanning microscopy and yellow fluorescent protein-tagged GWD3 CBM20 expressed in tobacco confirmed binding to starch granules in planta.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Phosphotransferases (Paired Acceptors)/metabolism , Starch/metabolism , Arabidopsis/cytology , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Aspergillus niger/enzymology , Aspergillus niger/genetics , Cyclodextrins/chemistry , Cytoplasmic Granules/genetics , Cytoplasmic Granules/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression , Glucan 1,4-alpha-Glucosidase/genetics , Glucan 1,4-alpha-Glucosidase/metabolism , Microscopy, Confocal/methods , Phosphotransferases (Paired Acceptors)/genetics , Protein Binding/physiology , Protein Structure, Tertiary/physiology , Starch/genetics , Structural Homology, Protein , Nicotiana/geneticsABSTRACT
Subsite affinity maps of long substrate binding clefts in barley alpha-amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3-12, revealed unfavorable binding energies at the internal subsites -3 and -5 and at subsites -8 and +3/+4 defining these subsites as binding barriers. Barley alpha-amylase 1 mutants Y105A and T212Y at subsite -6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (-2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in alpha-amylases.
Subject(s)
Hordeum/enzymology , Mutant Proteins/chemistry , Mutant Proteins/metabolism , alpha-Amylases/chemistry , alpha-Amylases/metabolism , Animals , Binding Sites , Biopolymers , Carbohydrate Conformation , Carbohydrate Sequence , Hordeum/genetics , Hydrolysis , Isoenzymes/chemistry , Isoenzymes/metabolism , Models, Molecular , Molecular Sequence Data , Mutant Proteins/genetics , Oligosaccharides/chemistry , Rabbits , Substrate Specificity , alpha-Amylases/geneticsABSTRACT
Until recently, the function of the fifth domain of the thermostable modular xylanase Xyn10A from Rhodothermus marinus was unresolved. A putative homologue to this domain was however identified in a mannanase (Man26A) from the same microorganism which raised questions regarding a common function. An extensive search of all accessible data-bases as well as the partially sequenced genomes of R. marinus and Cytophaga hutchinsonii showed that homologues of this domain were encoded by multiple genes in microorganisms in the phylum Bacteroidetes. Moreover, the domain occurred invariably at the C-termini of proteins that were predominantly extra-cellular/cell attached. A primary structure motif of three conserved regions including structurally important glycines and a proline was also identified suggesting a conserved 3D fold. This bioinformatic evidence suggested a possible role of this domain in mediating cell attachment. To confirm this theory, R. marinus was grown, and activity assays showed that the major part of the xylanase activity was connected to whole cells. Moreover, immunocytochemical detection using a Xyn10A-specific antibody proved presence of Xyn10A on the R. marinus cell surface. In the light of this, a revision of experimental data present on both Xyn10A and Man26A was performed, and the results all indicate a cell-anchoring role of the domain, suggesting that this domain represents a novel type of module that mediates cell attachment in proteins originating from members of the phylum Bacteroidetes.