Esterase Family Research Articles

Enzymatic acylation of flavonoid glycosides by a carbohydrate esterase of family 16.

The acetyl esterase of Trichoderma reesei belonging to carbohydrate esterase (CE) family 16 catalyzes transacylations to carbohydrate moieties of flavonoid glycosides, esculin and rutin. The enzyme recognizes as acyl donors vinyl esters of short carboxylic acids. Esculin was acylated at position 3 of the glucosyl residue in aqueous solutions saturated with vinyl acetate and vinyl propionate. The yields of esculin monoacetate and monopropionate of esculin in aqueous medium (esculin 40mM, enzyme 40µg/ml, 40°C, 3days) were 67 and 55%, respectively. Replacement of water by 2-propanol was required for a similar acylation of rutin at 4mM concentration. The yields of rutin monoacetate and propionate were 60 and 30%, respectively. The results indicate that the enzyme could be used for an easy modification of solubility and hydrophobicity of glycosylated compounds, including drugs and functional food additives.

Acetyl Xylan Esterase Axe1 (T. reesei, Carbohydrate Esterase Family 5) Supplemented to a (Hemi)cellulolytic Preparation Enhances Degradation of Recalcitrant Corn Silage Polysaccharides

The increase in hydrolytic activity towards corn silage water unextractable solids by supplementation of acetyl xylan esterase 1 (T. reesei, TrAxe1), belonging to arbohydrate esterase (CE) family 5, to an A. niger / T. emersonii enzyme preparation is presented. TrAxe1 was cloned and expressed in A niger. The hydrolytic activity of TrAxe1 was analysed with a p-nitrophenyl-acetate standard assay and by the measurement of the acetic acid released from neutral and acidic eucalyptus xylo-oligosaccharides. The activity obtained for TrAxe1 was compared to acetyl xylan esterases belonging to CE families 1, 5 and 16 from A. niger. High activities were obtained for the CE5 classified acetyl xylan esterases from T. reesei and A. niger. The data obtained confirm the suggested increase in hydrolytic activity by supplementation of CE5 classified acetyl xylan esterases to the (hemi)cellulolytic A. niger / T. emersonii preparation (Neumuller et al. 2014b)

Trichoderma longibrachiatum acetyl xylan esterase 1 enhances hemicellulolytic preparations to degrade corn silage polysaccharides.

Supplementation of a Trichoderma longibrachiatum preparation to an industrial Aspergillus niger/Talaromyces emersonii enzyme mixture demonstrated synergy for the saccharification of corn silage water-unextractable solids (WUS). Sub-fractions of the crude T. longibrachiatum preparation obtained after chromatography were analyzed regarding their hydrolytic activity. An acetyl xylan esterase 1 [Axe1, carbohydrate esterase (CE) family 5]-enriched sub-fraction closely mimicked the hydrolytic gain as obtained by supplementation of the complete, crude enzyme mixture (increase of 50%, 62% and 29% for Xyl, Ara and Glc, respectively). The acetic acid released from model polysaccharides (WUS) and oligosaccharides [neutral (AcXOS) and acidic (AcUXOS) xylo-oligosaccharides] by Axe1 was two and up to six times higher compared to the acetic acid released by acetyl xylan esterase A (AxeA, CE 1). Characterization of Axe1 treated AcXOS and AcUXOS revealed deacetylation of oligosaccharides that were not deacetylated by AxeA or the A. niger/T. emersonii preparation.

A Cold-Adapted Carbohydrate Esterase from the Oil-Degrading Marine Bacterium Microbulbifer thermotolerans DAU221: Gene Cloning, Purification, and Characterization

A cold-adapted carbohydrate esterase, CEST, belonging to the carbohydrate esterase family 6, was cloned from Microbulbifer thermotolerans DAU221. CEST was composed of 307 amino acids with the first 22 serving as a secretion signal peptide. The calculated molecular mass and isoelectric point of the mature enzyme were 31,244 Da and pH 5.89, respectively. The catalytic triad consisted of residues Ser37, Glu192, and His281 in the conserved regions: GQSNMXG, QGEX(D/N), and DXXH. The three-dimensional structure of CEST revealed that CEST belongs to the α/β-class of protein consisted of a central six-stranded β-sheet flanked by eight α-helices. The recombinant CEST was purified by His-tag affinity chromatography and the characterization showed its optimal temperature and pH were 15°C and 8.0, respectively. Specifically, CEST maintained up to 70% of its enzyme activity when preincubated at 50°C or 60°C for 6 h, and 89% of its enzyme activity when preincubated at 70°C for 1h . The results suggest CEST belongs to group 3 of the cold-adapted enzymes. The enzyme activity was increased by Na(+) and Mg(2+) ions but was strongly inhibited by Cu(+) and Hg(2+) ions, at all ion concentrations. Using p-nitrophenyl acetate as a substrate, the enzyme had a Km of 0.278 mM and a kcat of 1.9 s(-1). Site-directed mutagenesis indicated that the catalytic triad (Ser37, Glu192, and His281) and Asp278 were essential for the enzyme activity.

The biochemical characterization of the PDB entry 4EZI by in silico and in vitro quantitative techniques (777.2)

This study is part of the SBEVSL project, the purpose of which is to propose and confirm the function of uncharacterized protein structures, in the Protein Data Bank, by use of in silico and in vitro quantitative techniques. For over 3000 structures in the PDB, no function has been assigned. These structures were first screened with the PyMOL molecular graphics system, along with ProMOL plugin, which compares query structures against a library of enzyme active sites. The most promising alignments were further refined by sequence and structure comparison using BLAST and Dali. A structure of considerable interest is presented here: 4EZI. When 4EZI was queried with PyMOL/ ProMOL, the best alignment was with the dipeptidyl peptidase IV, 1ORV. After analysis with BLAST and Dali, the alignments revealed 4EZI matched closely to the esterase family, specifically 3H2H. This match was confirmed experimentally. The protein was purified using methods of transformation and column chromatography. Then, the predicted molecular weight was confirmed using SDS‐PAGE. Protein concentrations were determined by a BCA assay and enzymatic activity was confirmed using p‐nitrophenol esters of short‐chain carboxylic acids. In conclusion, 4EZI has been characterized as an esterase based both on in silico and in vitro quantitative methods. Funding was provided by RIT, Dowling College, and NIGMS 2R15GM078077‐02 & 3R15GM078077‐02S1.Grant Funding Source: Supported by NIGMS 2R15GM078077‐02, 3R15GM078077‐02S1

A Bacillus licheniformis pectin acetylesterase is specific for homogalacturonans acetylated at O-3

A recombinant acetylesterase from Bacillus licheniformis DSM13, belonging to carbohydrate esterase family 12, was purified and biochemically characterized. The purified enzyme, termed BliPAE, was capable of deacetylating acetylated pectins, e.g. sugar beet pectin (SBP). Contrary to its provisional annotation as rhamnogalacturonan acetylesterase, the enzyme specifically removed acetyl groups from the homogalacturonan region classifying it as a PAE. The recombinant enzyme has a molecular mass of 26.7kDa and shows optimal activity at pH 8.0 and 50°C. It is stable in the range pH 5.0–7.0 and below 50°C. Methylesterification of the galacturonic acid (GalA) moieties reduces the deacetylation efficacy of BliPAE. The enzyme efficiently removes acetyl groups from SBPs with low degree of methylesterification (DM) 9-30, releasing about 75% of the acetyl groups present in the homogalacturonan. Furthermore, 1H NMR of polymer and LC-HILIC-MSn after endo-PGII and PL degradation were used to structurally characterize the BliPAE-modified pectins. The results show that BliPAE removes acetyl groups specifically when substituted at the O-3 position of GalA moieties.

A unique octameric structure of Axe2, an intracellular acetyl-xylooligosaccharide esterase fromGeobacillus stearothermophilus

Geobacillus stearothermophilus T6 is a thermophilic, Gram-positive soil bacterium that possesses an extensive and highly regulated hemicellulolytic system, allowing the bacterium to efficiently degrade high-molecular-weight polysaccharides such as xylan, arabinan and galactan. As part of the xylan-degradation system, the bacterium uses a number of side-chain-cleaving enzymes, one of which is Axe2, a 219-amino-acid intracellular serine acetylxylan esterase that removes acetyl side groups from xylooligosaccharides. Bioinformatic analyses suggest that Axe2 belongs to the lipase GDSL family and represents a new family of carbohydrate esterases. In the current study, the detailed three-dimensional structure of Axe2 is reported, as determined by X-ray crystallography. The structure of the selenomethionine derivative Axe2-Se was initially determined by single-wavelength anomalous diffraction techniques at 1.70 Å resolution and was used for the structure determination of wild-type Axe2 (Axe2-WT) and the catalytic mutant Axe2-S15A at 1.85 and 1.90 Å resolution, respectively. These structures demonstrate that the three-dimensional structure of the Axe2 monomer generally corresponds to the SGNH hydrolase fold, consisting of five central parallel β-sheets flanked by two layers of helices (eight α-helices and five 310-helices). The catalytic triad residues, Ser15, His194 and Asp191, are lined up along a substrate channel situated on the concave surface of the monomer. Interestingly, the Axe2 monomers are assembled as a `doughnut-shaped' homo-octamer, presenting a unique quaternary structure built of two staggered tetrameric rings. The eight active sites are organized in four closely situated pairs, which face the relatively wide internal cavity. The biological relevance of this octameric structure is supported by independent results obtained from gel-filtration, TEM and SAXS experiments. These data and their comparison to the structural data of related hydrolases are used for a more general discussion focusing on the structure-function relationships of enzymes of this category.

Bioinformatic characterisation of genes encoding cell wall degrading enzymes in the Phytophthora parasitica genome.

BackgroundA critical aspect of plant infection by the majority of pathogens is penetration of the plant cell wall. This process requires the production and secretion of a broad spectrum of pathogen enzymes that target and degrade the many complex polysaccharides in the plant cell wall. As a necessary framework for a study of the expression of cell wall degrading enzymes (CWDEs) produced by the broad host range phytopathogen, Phytophthora parasitica, we have conducted an in-depth bioinformatics analysis of the entire complement of genes encoding CWDEs in this pathogen’s genome.ResultsOur bioinformatic analysis indicates that 431 (2%) of the 20,825 predicted proteins encoded by the P. parasitica genome, are carbohydrate-active enzymes (CAZymes) involved in the degradation of cell wall polysaccharides. Of the 431 proteins, 337 contain classical N-terminal secretion signals and 67 are predicted to be targeted to the non-classical secretion pathway. Identification of CAZyme catalytic activity based on primary protein sequence is difficult, nevertheless, detailed comparisons with previously characterized enzymes has allowed us to determine likely enzyme activities and targeted substrates for many of the P. parasitica CWDEs. Some proteins (12%) contain more than one CAZyme module but, in most cases, multiple modules are from the same CAZyme family. Only 12 P. parasitica CWDEs contain both catalytically-active (glycosyl hydrolase) and non-catalytic (carbohydrate binding) modules, a situation that contrasts with that in fungal phytopathogens. Other striking differences between the complements of CWDEs in P. parasitica and fungal phytopathogens are seen in the CAZyme families that target cellulose, pectins or β-1,3-glucans (e.g. callose). About 25% of P. parasitica CAZymes are solely directed towards pectin degradation, with the majority coming from pectin lyase or carbohydrate esterase families. Fungal phytopathogens typically contain less than half the numbers of these CAZymes. The P. parasitica genome, like that of other Oomycetes, is rich in CAZymes that target β-1,3-glucans.ConclusionsThis detailed analysis of the full complement of P. parasitica cell wall degrading enzymes provides a framework for an in-depth study of patterns of expression of these pathogen genes during plant infection and the induction or repression of expression by selected substrates.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-785) contains supplementary material, which is available to authorized users.

Characterization and Expression of a GDSL-Like Lipase Gene from Brassica napus in Nicotiana benthamiana

The GDSL esterase and lipase families play important roles in abiotic stress, pathogen defense, seed development and lipid metabolism. Identifying the lipase activity of the putative GDSL lipase is the prerequisite for dissecting its function. According to the sequence similarity and the conserved domains, we cloned the Brassica napus BnGLIP gene, which encodes a GDSL-like protein. We failed to identify the BnGLIP lipase activity in the bacterium and yeast expression systems. In this paper, we expressed the BnGLIP gene by fusing a 6× His tag in Nicotiana benthamiana and purified the recombinant protein. The extraction buffer contained 1 % (v/v) n-caprylic acid and was able to remove most of the protein impurities. About 50 μg of recombinant BnGLIP was obtained from 1 g of N. benthamiana leaves. The lipase activity was tested with the purified BnGLIP and the maximum enzyme activity reached 17.7 mM/mg. In conclusion, this study found that the recombinant protein BnGLIP expressed in tobacco system was effectively purified and was detected as a GDSL lipase.

Characterization of a novel cold-active esterase isolated from swamp sediment metagenome

A functional screen of a metagenomic library from "Upo" swamp sediment in Korea identified a gene EstL28, the product of which displayed lipolytic properties on a tributyrin-supplemented medium. The EstL28 sequence encodes a 290 amino acid protein (designated as EstL28), with a predicted molecular weight of 31.3 kDa. The encoded EstL28 protein exhibited the highest sequence similarity (45 %) to a hydrolase found in Streptococcus sanguinis. Phylogenetic analysis indicated that EstL28 belongs to a currently uncharacterized family of esterases. Within the conserved α/β-hydrolase 6 domain, the EstL28 retains the catalytic triad Ser₁₀₃-Asp₂₄₈-His₂₆₈ that is typical of esterases. The Ser103 residue in the catalytic triad is located in the consensus pentapeptide motif GXSXG. The purified EstL28 enzyme worked optimally at 35 °C and pH 8.5 and remained stable at temperatures lower than 20 °C. The catalytic activity of EstL28 was maximal with p-nitrophenyl butyrate, indicating that it was an esterase. This enzyme also exhibited stable activity in the presence of methanol, ethanol, isopropanol, and dimethyl sulfoxide. Therefore, the level of stability in organic solvents and cold temperature suggests that EstL28 has potential for many biotechnological applications.

Distinct roles of carbohydrate esterase family CE16 acetyl esterases and polymer-acting acetyl xylan esterases in xylan deacetylation

Mass spectrometric analysis was used to compare the roles of two acetyl esterases (AE, carbohydrate esterase family CE16) and three acetyl xylan esterases (AXE, families CE1 and CE5) in deacetylation of natural substrates, neutral (linear) and 4-O-methyl glucuronic acid (MeGlcA) substituted xylooligosaccharides (XOS). AEs were similarly restricted in their action and apparently removed in most cases only one acetyl group from the non-reducing end of XOS, acting as exo-deacetylases. In contrast, AXEs completely deacetylated longer neutral XOS but had difficulties with the shorter ones. Complete deacetylation of neutral XOS was obtained after the combined action of AEs and AXEs. MeGlcA substituents partially restricted the action of both types of esterases and the remaining acidic XOS were mainly substituted with one MeGlcA and one acetyl group, supposedly on the same xylopyranosyl residue. These resisting structures were degraded to great extent only after inclusion of α-glucuronidase, which acted with the esterases in a synergistic manner. When used together with xylan backbone degrading endoxylanase and β-xylosidase, both AE and AXE enhanced the hydrolysis of complex XOS equally.

Characterization of a novel thermophilic pyrethroid-hydrolyzing carboxylesterase from Sulfolobus tokodaii into a new family

Abstract A novel gene ST2026 encoding a putative carboxylesterase from the thermophilic crenarchaeota Sulfolobus tokodaii (named EstSt7) was cloned and functionally overexpressed in Escherichia coli. The recombinant enzyme was purified to homogeneity after heat treatment, Ni-NTA affinity and Superdex-200 gel filtration chromatography. EstSt7 showed maximum activity at 80 °C over 30 min and had a half-life of 180 min at 90 °C. Its enzymatic activity was stable in the pH range of 8.0–10.0 with an optimum at 9.0. The enzyme exhibited significant esterase activity toward various p-nitrophenyl esters and the most preferable substrate was p-nitrophenyl butyrate (kcat/Km of 246.3 s−1 mM−1). In addition, EstSt7 showed high activity and stability against organic solvents (20% and 50% v/v) and detergents (1% and 5% v/v). Furthermore, EstSt7 could efficiently hydrolyze a wide range of synthetic pyrethroids including fenpropathrin, permethrin, cypermethrin, cyhalothrin, deltamethrin and bifenthrin, which makes it a potential candidate for the detoxification of pyrethroids for the purpose of biodegradation. Sequence alignment, phylogenetic analysis and comparison of the conserved motif reveal that this novel carboxylesterase EstSt7 should be grouped into a new bacterial lipase and esterase family.

Mode of action of acetylxylan esterases on acetyl glucuronoxylan and acetylated oligosaccharides generated by a GH10 endoxylanase

BackgroundSubstitutions on the xylan main chain are widely accepted to limit plant cell wall degradability and acetylations are considered as one of the most important obstacles. Hence, understanding the modes of action of a range of acetylxylan esterases (AcXEs) is of ample importance not only to increase the understanding of the enzymology of plant decay/bioremediation but also to enable efficient bioconversion of plant biomass. MethodsIn this study, the modes of action of acetylxylan esterases (AcXEs) belonging to carbohydrate esterase (CE) families 1, 4, 5 and 6 on xylooligosaccharides generated from hardwood acetyl glucuronoxylan were compared using MALDI ToF MS. Supporting data were obtained by following enzymatic deacetylation by 1H NMR spectroscopy. ConclusionsNone of the used enzymes were capable of complete deacetylation, except from linear xylooligosaccharides which were completely deacetylated by some of the esterases in the presence of endoxylanase. A clear difference was observed between the performance of the serine-type esterases of CE families 1, 5 and 6, and the aspartate-metalloesterases of family CE4. The difference is mainly due to the inability of CE4 AcXEs to catalyze deacetylation of 2,3-di-O-acetylated xylopyranosyl residues. Complete deacetylation of a hardwood acetyl glucuronoxylan requires additional deacetylating enzyme(s). General significanceThe results contribute to the understanding of microbial degradation of plant biomass and outline the way to achieve complete saccharification of plant hemicelluloses which did not undergo alkaline pretreatment.

Structural and functional characterisation of TesA - a novel lysophospholipase A from Pseudomonas aeruginosa.

TesA from Pseudomonas aeruginosa belongs to the GDSL hydrolase family of serine esterases and lipases that possess a broad substrate- and regiospecificity. It shows high sequence homology to TAP, a multifunctional enzyme from Escherichia coli exhibiting thioesterase, lysophospholipase A, protease and arylesterase activities. Recently, we demonstrated high arylesterase activity for TesA, but only minor thioesterase and no protease activity. Here, we present a comparative analysis of TesA and TAP at the structural, biochemical and physiological levels. The crystal structure of TesA was determined at 1.9 Å and structural differences were identified, providing a possible explanation for the differences in substrate specificities. The comparison of TesA with other GDSL-hydrolase structures revealed that the flexibility of active-site loops significantly affects their substrate specificity. This assumption was tested using a rational approach: we have engineered the putative coenzyme A thioester binding site of E. coli TAP into TesA of P. aeruginosa by introducing mutations D17S and L162R. This TesA variant showed increased thioesterase activity comparable to that of TAP. TesA is the first lysophospholipase A described for the opportunistic human pathogen P. aeruginosa. The enzyme is localized in the periplasm and may exert important functions in the homeostasis of phospholipids or detoxification of lysophospholipids.

A novel cephalosporin deacetylating acetyl xylan esterase from Bacillus subtilis with high activity toward cephalosporin C and 7-aminocephalosporanic acid

A cephalosporin deacetylating acetyl xylan esterase was cloned from the genomic DNA of Bacillus subtilis CICC 20034 and functionally expressed in Escherichia coli. Its gene contained an open reading frame of 957 bp encoding 318 amino acids with a calculated mass of 35,607 Da, and it displayed significant identity to acetyl xylan esterases from Bacillus sp. 916, B. subtilis 168, and Bacillus pumilus Cect5072. The enzyme was a native homohexamer but a trimer under the condition of 1% sodium dodecyl sulfate (SDS); both forms were active and could transit to each other by incubating in or removing SDS. The enzyme belongs to carbohydrate esterase family 7 and had a double specificity on both the acetylated oligosaccharide and cephalosporin C (CPC) and 7-aminocephalosporanic acid (7-ACA). The activity of this purified enzyme toward CPC and 7-ACA was highest among all the acetyl xylan esterase from CE family 7, which were 484 and 888 U/mg, respectively, and endowed itself with great industrial interest on semi-synthetic β-lactam antibiotics. The optimum pH of the purified enzyme was 8.0, and the optimum temperature was 50 °C, and the enzyme had high thermal stability, broad range of pH tolerance, and extremely organic solvent tolerance.

Functional characterisation of a metagenome derived family VIII esterase with a deacetylation activity on β-lactam antibiotics

Family VIII esterases represent a poorly characterised esterase family, with high sequence identity to class C β-lactamases, peptidases and penicillin binding proteins. This study reports on the metagenomic library screening and biochemical characterisation of a novel esterase (Est22) derived from an acidic Leachate environment. The enzyme is 423 amino acids in length and contained 22 aa signal peptide. The Est22 primary structure revealed the presence of N-terminus S-x-x-K sequence, which is also highly conserved in class C β-lactamases, peptidases as well as carboxylesterases belonging to family VIII. Phylogenetic analysis using the representative sequences from class C β-lactamases and family VIII esterases indicated that Est22 is a member of family VIII esterases. Substrate specificity profiling using p-nitrophenyl esters (C2–C16) indicated that Est22 preferred shorter chain p-nitrophenyl esters (C2–C5), a characteristic that is typical for true carboxylesterases. In addition of hydrolysing Nitrocefin, Est22 also hydrolysed first generation cephalosporin based derivatives. Detailed selectivity study using different cephalosporin based substrates indicated that Est22 selectively hydrolyse the ester bond of a cephalosporin derivatives leaving the amide bond of the β-lactam ring intact. The selective nature of Est22 makes this enzyme a potential candidate for the use in the synthesis and modification cephalosporin based molecules.

Crystal Structure of Tannase from Lactobacillusplantarum

Tannins are water-soluble polyphenolic compounds in plants. Hydrolyzable tannins are derivatives of gallic acid (3,4,5-trihydroxybenzoic acid) or its meta-depsidic forms that are esterified to polyol, catechin, or triterpenoid units. Tannases are a family of esterases that catalyze the hydrolysis of the galloyl ester bond in hydrolyzable tannins to release gallic acid. The enzymes have found wide applications in food, feed, beverage, pharmaceutical, and chemical industries since their discovery more than a century ago, although little is known about them at the molecular level, including the details of the catalytic and substrate binding sites. Here, we report the first three-dimensional structure of a tannase from Lactobacillus plantarum. The enzyme displays an α/β structure, featured by a large cap domain inserted into the classical serine hydrolase fold. A catalytic triad was identified in the structure, which is composed of Ser163, His451, and Asp419. During the binding of gallic acid, the carboxyl group of the molecule forges hydrogen-bonding interactions with the catalytic triad of the enzyme while the three hydroxyl groups make contacts with Asp421, Lys343, and Glu357 to form another hydrogen-bonding network. Mutagenesis studies demonstrated that these residues are indispensable for the activity of the enzyme. Structural studies of the enzyme in complex with a number of substrates indicated that the interactions at the galloyl binding site are the determinant force for the binding of substrates. The single galloyl binding site is responsible for the esterase and depsidase activities of the enzyme.

Characterization of three new carboxylic ester hydrolases isolated by functional screening of a forest soil metagenomic library

Three new lipolytic genes were isolated from a forest soil metagenomic library by functional screening on tributyrin agar plates. The genes SBLip1, SBLip2 and SBLip5.1 respectively encode polypeptides of 445, 346 and 316 amino acids. Phylogenetic analyses revealed that SBLip2 and SBLip5.1 belong to bacterial esterase/lipase family IV, whereas SBLip1 shows similarity to class C β-lactamases and is thus related to esterase family VIII. The corresponding genes were overexpressed and their products purified by affinity chromatography for characterization. Analyses of substrate specificity with different p-nitrophenyl esters showed that all three enzymes have a preference for short-acyl-chain p-nitrophenyl esters, a feature of carboxylesterases as opposed to lipases. The β-lactamase activity of SBLip1, measured with the chromogenic substrate nitrocefin, was very low. The three esterases have the same optimal pH (pH 10) and remain active across a relatively broad pH range, displaying more than 60% activity between pH 6 and 10. The temperature optima determined were 35°C for SBLip1, 45°C for SBLip2 and 50°C for SBLip5.1. The three esterases displayed different levels of tolerance to salts, solvents and detergents, SBLip2 being overall more tolerant to high concentrations of solvent and SBLip5.1 less affected by detergents.

Positional specifity of acetylxylan esterases on natural polysaccharide: An NMR study

BackgroundMicrobial degradation of acetylated plant hemicelluloses involves besides enzymes cleaving the glycosidic linkages also deacetylating enzymes. A detailed knowledge of the mode of action of these enzymes is important in view of the development of efficient bioconversion of plant materials that did not undergo alkaline pretreatment leading to hydrolysis of ester linkages. MethodsIn this work deacetylation of hardwood acetylglucuronoxylan by acetylxylan esterases from Streptomyces lividans (carbohydrate esterase family 4) and Orpinomyces sp. (carbohydrate esterase family 6) was monitored by 1H-NMR spectroscopy. ResultsThe 1H-NMR resonances of all acetyl groups in the polysaccharide were fully assigned. The targets of both enzymes are 2- and 3-monoacetylated xylopyranosyl residues and, in the case of the Orpinomyces sp. enzyme, also the 2,3-di-O-acetylated xylopyranosyl residues. Both enzymes do not recognize as a substrate the 3-O-acetyl group on xylopyranosyl residues α-1,2-substituted with 4-O-methyl-d-glucuronic acid. ConclusionsThe 1H-NMR spectroscopy approach to study positional and substrate specificity of AcXEs outlined in this work appears to be a simple way to characterize catalytic properties of enzymes belonging to various CE families. SignificanceThe results contribute to development of efficient and environmentally friendly procedures for enzymatic degradation of plant biomass.

Distinct functions of polysaccharide deacetylases in cell shape, neutral polysaccharide synthesis and virulence of Bacillus anthracis

Peptidoglycan deacetylases (PGNG-dacs) belong to the Carbohydrate Esterase Family 4 (CE4) and have been described as required for bacterial evasion to lysozyme and innate immune responses. Interestingly, there is an unusual occurrence of 10 putative polysaccharide deacetylases, including five PGNG-dacs, in the Bacillus sp. genomes, especially B. cereus and B. anthracis. To elucidate the physiological role of these multiple deacetylases, we employed genetic analysis and protein localization studies of five putative PGNG-dacs from B. anthracis as well as biochemical analysis of their corresponding homologues from B. cereus. Our data confirm that three enzymes are PGNG-dacs. While BA1977, associated with lateral peptidoglycan synthesis, is a bona fide peptidoglycan deacetylase involved in resistance to host lysozyme and required for full virulence, BA1961 and BA3679 participate in the biogenesis of the peptidoglycan during both elongation and cell division. Furthermore, two enzymes are important for neutral polysaccharide attachment to PG and consequently anchoring of S-layer proteins (BA5436) and for polysaccharide modification (BA2944). Our results provide novel and fundamental insights into the function of polysaccharide deacetylases in a major bioterrorism agent.