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

Biochimica et Biophysica Acta 1830 (2013) 5075–5086

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Mode of action of acetylxylan esterases on acetyl glucuronoxylan and acetylated oligosaccharides generated by a GH10 endoxylanase Peter Biely a,⁎, Mária Cziszárová a, Iveta Uhliariková a, Jane W. Agger b, Xin-Liang Li c, Vincent G.H. Eijsink b, Bjorge Westereng b,d a

Institute of Chemistry, Center of Glycomics, Slovak Academy of Sciences, Dúbravská cesta 9, 84538 Bratislava, Slovakia Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Aas, Norway Youtell Biotech Inc., Bothell, WA, USA d University of Copenhagen, Faculty of Science, Rolighedsvej 23, 1958 Frederiksberg C, Denmark b c

a r t i c l e

i n f o

Article history: Received 15 May 2013 Received in revised form 15 July 2013 Accepted 17 July 2013 Available online 24 July 2013 Keywords: Acetylxylan esterase Carbohydrate esterase family Positional specificity Acetyl glucuronoxylan MALDI ToF MS NMR

a b s t r a c t Background: Substitutions 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. Methods: In 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. Conclusions: None 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 significance: The 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. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Hardwood glucuronoxylan and cereal arabinoxylans are partially acetylated [1–3]. Consequently, all procedures for biorefining of plant biomass that do not involve alkaline pretreatments will generate partially

Abbreviations: AcXE, acetylxylan esterase; CE, carbohydrate esterase; Xylp, or D-xylopyranosyl; Xyl2–Xyl7, β-1,4-xylobiose–β-1,4-xyloheptaose; MeGlcA, 4-O-methyl-D-glucuronic acid or 4-O-methyl-D-glucuronosyl; MeGlcA3Xyl3, 4 - O - methyl - D-glucuronosyl–α - 1,2-D -xylopyranosyl–β-1,4-xylopyranosyl–β-1,4xylopyranose (the upper index indicates the number of the xylosyl residues from the reducing end substituted with MeGlcA); XylxAcy, acetylated β-1,4-xylooligosaccharide containing x xylose residues and y acetyl groups; MeGlcAXylxAcy, acetylated aldouronic acid containing one MeGlcA, x xylose residues and y acetyl groups; HexXylxAcy, oligosaccharide containing one hexopyranose residue of unknown nature, x xylose residues and y acetyl groups; UXylxAcy, oligosaccharide containing an unknown component U, x xylose residues and y acetyl groups ⁎ Corresponding author. Tel.: +421 2 59410275; fax: +421 2 59410222. E-mail address: [email protected] (P. Biely). D-xylopyranose

0304-4165/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbagen.2013.07.018

acetylated hemicellulose fractions. Complete hydrolysis of, e.g. hardwood acetyl glucuronoxylan, not only requires glycoside hydrolases (endo-β1,4-xylanases, α-glucuronidases, β-xylosidases) but also deacetylating enzymes, called acetylxylan esterases (AcXEs) [4]. The role of the deacetylating enzymes is to create new sites for productive binding of glycoside hydrolases, thus enabling complete hydrolysis. Deacetylation is complicated by considerable variation regarding the position of the acetyl group and substitution of the vicinal hydroxyl group (Fig. 1). A major portion of Xylp residues is monoacetylated at position 2 or 3, or 2,3-di-Oacetylated. 3-O-acetylation frequently accompanies the substitution of Xylp residues with MeGlcA [5–7]. So far, AcXEs have been found in four of the carbohydrate esterase (CE) families defined in the CAZy database [8], namely families 1, 4, 5 and 6 [4]. Which esterases are needed to achieve complete deacetylation of xylan poly- or oligosaccharides still remains to be elucidated and there is a clear need for studying the action of various AcXEs on natural substrates. So far, literature data are scarce. AcXEs of Chrysosporium lucknowense belonging to CE families 1 and 5 were found to be capable

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O

R O O O

OH CH3

O

O HO

O O

O HO

O O OH

CH3

H3C HO H3CO

O O

O

O O

OH

O

O O

O

R1

O CH3 O

CH3

O COOH

Fig. 1. Four types of acetylation of Xylp residues in hardwood acetyl glucuronoxylans. From the left: 3-O-acetyl-, 2-O-acetyl-, 3-O-acetyl-2-O-MeGlcA- and 2,3-di-O-acetyl-Xylp.

of removing acetyl groups from both positions 2 and 3 of linear xylooligosaccharides [9]. This conclusion was based on monitoring the deacetylation of linear oligosaccharides by MALDI ToF MS in combination with capillary electrophoresis of derivatized products [9]. The mode of action of Streptomyces lividans CE4 and Orpinomyces sp. CE6 AcXEs on birchwood acetyl glucuronoxylan has been studied by 1H NMR spectroscopy [10]. The release of individual acetyl groups was monitored by measuring two sets of signals, one in the anomeric region of the spectrum, and the other with the signals of the acetyl methyl groups [5–7,10]. The targets of both enzymes were 2- and 3-O-monoacetylated Xylp residues. The Orpinomyces sp. esterase also attacked the 2,3-di-O-acetylated Xylp residues [10]. Both enzymes ignored the 3-O-acetyl group on Xylp residues that were α-1,2-substituted with MeGlcA. It is interesting that AcXEs, in general, showed dual positional specificity on acetylated methyl glycosides [11–13], but strong selectivity for position 2 on monoacetyl derivatives of 4-nitrophenyl glycosides [4,14]. Taking together the current status of knowledge and the increasing need for controlled and efficient conversion of hemicelluloses, the following pertinent questions arise: i) How do the various AcXEs, belonging to different CE families, perform on acetyl glucuronoxylan in the presence or absence of endo-β-1,4-xylanases?, ii) Will endoxylanase cleavage of the xylan main chain make all acetyl groups accessible to esterases?, iii) What is the mechanism allowing some of the enzymes to deacetylate two different positions on Xylp residues? and iv) Is the apparent positional flexibility of some of the enzymes due to migration of the acetyl group between positions 2 and 3? To answer these questions and to provide a view on AcXE variability, we have studied the actions of enzymes belonging to CE families 1, 4, 5 and 6 on acetylated xylooligosaccharides generated from aspen acetyl glucuronoxylan by a GH10 endoxylanase. We found that the NMR technique introduced for studying AcXE mode of action on a polymeric substrate [10] cannot be applied to a mixture of acetylated xylooligosaccharides due to excessively complex spectra. Therefore, MALDI ToF MS was adopted for product analysis. After resolving the acetylated xylooligosaccharides in the starting material, they were treated with different AcXEs in the absence or presence of an endo-xylanase and changes in the degree of acetylation were monitored. The NMR approach was used for monitoring the rates of specific deacetylation processes for those enzymes that had not previously been studied in this manner. Apart from illustrating differences between the various AcXE types, the results confirmed the interplay between xylanases and esterases. Maximum deacetylation required the presence of an active endoxylanase further degrading the partially deacetylated xylooligosaccharides. Complete deacetylation of all oligosaccharides could not be achieved. The resistant acetyl groups have been tentatively identified based on knowledge of: i) the architecture of GH10 xylanases, ii) the known catalytic properties of AcXEs on artificial substrates such as acetylated glycosides, and iii) the positional specificity of the esterases on polymeric substrates as demonstrated by 1 H NMR spectroscopy. It appears that xylooligosaccharides contain additional resistant acetyl groups which are not attacked by the typical AcXEs. The combination of existing and newly generated data discussed below shows that complete removal of the acetyl groups from acetyl glucuronoxylan requires deacetylases with specificities that have not yet been detected.

2. Materials and methods 2.1. Polysaccharides Birchwood acetyl glucuronoxylan prepared by steam explosion of birchwood saw dust was supplied by Dr. Henry Schneider (NRCC, Ottawa, Canada). Aspen acetyl glucuronoxylan with an approximate mass distribution from 500 Da to 2500 Da (roughly estimated from MALDI-TOF MS analyses) was prepared by steam explosion (at 200 °C for 10 min) of milled aspen wood (milled on a Retsch mill with a 500 μm sieve) using the experimental setup described by Horn et al. (2011) [15]. The resulting soluble material was purified and concentrated by ultrafiltration on a tangential flow ultrafiltration unit (Pellicon, Millipore) collecting the permeate in the first filtration step (10 kDa cut-off) and the retentate in a subsequent filtration with a 1 kDa cut-off membrane. The xylan containing retentate was collected and lyophilized. 2.2. Enzymes Endo-β-1,4-xylanase of GH10 family from Clostridium thermocellum (Xyn Z or CtGH10) was a recombinant enzyme prepared as described below. A DNA sequence coding for residues 511 to 837 of CtGH10 [16] was synthesized. To facilitate cloning of the sequence into pET-24b(−) (Invitrogen Corp., Carlsbad, CA), BamHI and XhoI sites were added on the 5′ and 3′ ends, respectively, leading to a total fragment size of 991 bp, which was synthesized by GenScript Corp. (Piscataway, NJ). After cloning of the gene into pET-24b(−) and verification of the sequence by sequencing with T7 promoter and terminator oligonucleotides as primers, Escherichia coli HMS 174 (DE3) competent cells were transformed with the plasmid and four primary clones were analyzed for xylanase production after induction in liquid culture and disruption of the cells. The sonicated supernatant of all the four cultures displayed activity against birchwood xylan. One liter culture of the highest xylanase-producing clone was prepared, and a clarified cell-free supernatant was subject to purification using a prepacked nickel affinity column (GE Life Science USA, Piscataway, NJ, USA) controlled by an AKTAbasic System (GE). The 35-kDa His-tagged xylanase was further purified by anion exchange chromatography with a MonoQ HR 16/10 column (GE) using 50 mM Tris/HCl, pH 7.5 for binding; proteins were eluted by a gradient of 0–0.5 M NaCl in 50 mM Tris/HCl, pH 7.5. The flow rate was 3.0 ml/min and fractions of 1.5 ml each were collected. Fractions containing a high level of xylanase activity were pooled and concentrated using a Centricon Plus tube (Millipore Co., Billerica, MA, USA). The CE1 AcXE from Schizophyllum commune (ScCE1) was purified as described [11]. Purified CE4 AcXE from S. lividans (SlCE4) [17] was kindly supplied by Drs. Claude Dupont and Dieter Kluepfel (Institute of Armand Frappier, Laval, Canada). A similar enzyme from C. thermocellum (CtCE4) [18] was provided by Profs. Carlos M.G.A. Fontes (Universidade Técnica de Lisboa, Portugal) and Gideon J. Davies (University of York, UK). A purified CE5 AcXE from Trichoderma reesei (TrCE5) [19] was kindly provided by Prof. Maija Tenkanen (University of Helsinki, Finland). A recombinant CE6 AcXE from Orpinomyces sp. (OCE6) was from Megazyme Int. (Ireland).

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2.3. Enzymatic hydrolysis of aspen acetyl glucuronoxylan A solution of aspen acetyl glucuronoxylan (0.5%, w/v) in 0.05 M Tris/ HCl buffer (pH 6.5) was incubated with the recombinantly produced catalytic module of CtGH10 [16] (0.07 mg/ml) at 40 °C. After 24 h of incubation, part of the reaction mixture was heated for 5 min at 100 °C to denature the enzyme. 50 μl aliquots of the reaction mixture with active or heat-inactivated xylanase were then mixed with small volumes of solutions of AcXEs to give the following final enzyme concentrations: ScCE1, 0.13 mg/ml; SlCE4, 0.045 mg/ml; CtCE4, 0.4 mg/ml; TrCE5, 0.13 mg/ml and OCE6, 0.12 mg/ml. The mixtures were then incubated for another 24 h at 40 °C. After denaturation of the enzymes (5 min, 100 °C), the samples were cooled, the product pattern was examined by TLC on silica gels (Merck, Germany), and the samples were dried in vacuum. TLC was done in ethyl acetate–acetic acid–2-propanol–water (2:1:0.1, v/v) or ethyl acetate–acetic acid–2-propanol–formic acid–water (25:10:5:1:15, v/v) and the sugars were detected with N-(1-naphthyl)-ethylenediamine dihydrochloride reagent [20]. This procedure yielded samples suitable for MALDI ToF MS analysis as described below. 2.4. MALDI ToF MS Two μl of a 9 mg/ml mixture of 2,5-dihydroxybenzoic acid (DHB) in 30% acetonitrile was applied to an MTP 384 ground steel target plate TF (Bruker Daltonics). One μl sample was then mixed into the DHB droplet and dried under a stream of air. The samples were analyzed with an Ultraflex MALDI-TOF/TOF instrument (Bruker Daltonics GmbH, Bremen, Germany) equipped with a nitrogen 337 nm laser beam. The instrument was operated in positive acquisition mode and controlled by the FlexControl 3.3 software package. All spectra were obtained using the reflectron mode with an acceleration voltage of 25 kV, a reflector voltage of 26, and pulsed ion extraction of 40 ns in the positive ion mode. The acquisition range used was from m/z 0 to 7000. The data was collected from averaging 400 laser shots, with the lowest laser energy necessary to obtain sufficient signal to noise ratios. Peak lists were generated from the MS spectra using Bruker flexAnalysis software (version 3.3). 2.5. Monitoring of the AcXE action on acetylated polysaccharide by 1H NMR spectroscopy The positional specificity of the AcXEs from S. commune (ScCE1) and C. thermocellum (CtCE4) was examined by 1H NMR spectroscopy using birch acetyl glucuronoxylan as described before [10]. 10 mg of polysaccharide dried twice from D2O was dissolved in 0.65 ml D2O and the pH of the solution was adjusted to 6.0 with a 0.2 M solution of deuterized sodium acetate (Aldrich Chemicals, USA) in D2O, yielding a final substrate concentration of 1.5%. After recording the 1H NMR spectrum of the starting polysaccharide, AcXEs lyophilized twice from D2O were added. The concentration of the ScCE1 and CtCE4 enzyme in the reaction mixtures was 0.065 mg/ml and 0.4 mg/ml, respectively. The changes in signal intensity of the acetyl groups of glucuronoxylan were monitored over time at 25 °C using a VNMRS 400 MHz Varian spectrometer equipped with a 5 mm 1H–19F/15N–31P PFG AutoX DB NB probe head. The experimental details of the NMR measurements and the assignment of acetyl group signals in the anomeric region and in the region of acetyl methyl groups have been described in detail by Uhliariková et al. [10]. 3. Results 3.1. Fragments of acetyl glucuronoxylan generated by GH10 xylanase The mode of action of AcXEs was examined on a mixture of oligosaccharides generated from aspen acetyl glucuronoxylan by a GH10 xylanase from C. thermocellum. After thermal denaturation of the endoxylanase, the starting mixture was analyzed by MALDI ToF MS. Products released correspond to acetylated xylooligosaccharides and acetylated

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aldouronic acids of various lengths (Fig. 2A, and Table 1). These results are in agreement with previously published data showing a high frequency of acetylation for aspen, birch and eucalyptus glucuronoxylan extracted in the absence of alkali [5–7,21,22]. Acidic oligosaccharides regularly occurred as pairs of ions, comprising a sodium adduct ion of the general formula [MeGlcAXylxAcy + Na]+ and the corresponding sodium salt [MeGlcAXylxAcy-H + 2Na]+ (+m/z 22), as is frequently observed for acidic carbohydrates [23]. For the sake of simplicity, such double adducts are not tabulated but labeled in Fig. 2 by asterisks. A peculiar series of ions was observed that contained, in addition to xylose residues and acetyl groups, an unknown component designated as U (m/z 252). The sodium adducts of this series of the general formula [UXylxAcy + Na]+ (see Fig. 2A) were: U 275, UAc 317, UXyl 408, UXylAc 450, UXyl2 540, UXyl2Ac 582, UXyl2Ac2 624, and UXyl3Ac2 756. These ions were more visible in the oligosaccharide mixtures treated with AcXEs (Fig. 2B–E; see below). All observed neutral xylooligosaccharides generated by the xylanase were acetylated, starting from the mono- or di-O-acetyl derivatives up to derivatives with the same number of acetyl groups as the number of xylosyl residues (Table 1). The abundance of ions representing neutral xylooligosaccharides decreased dramatically in going from xylohexaose to larger xylooligosaccharides. The only neutral non-acetylated sugar detected was xylobiose. All observed acidic xylooligosaccharides, i.e. MeGlcA containing aldouronic acids, were acetylated. The shortest one, an aldotriouronic acid, MeGlcAXyl2, was released as a monoacetyl derivative. Higher aldouronic acids, such as MeGlcAXyl3 and MeGlcAXyl4, carried varying numbers of acetyl groups, and included minor amounts of species with an equal amount of Xyl and acetyl groups (Table 1). The detected species included longer acetylated aldouronic acids such as MeGlcAXyl8Ac5, MeGlcAXyl8Ac6, MeGlcAXyl8Ac7, MeGlcAXyl9Ac6, MeGlcAXyl9Ac7, MeGlcAXyl9Ac8, MeGlcAXyl10Ac7, MeGlcAXyl10Ac8 and MeGlcAXyl10Ac9. These longer species and their partially deacetylated forms are visible in Fig. 2A, D and E, however they are not listed in Tables 1 and 2 to keep the size of both tables to fit in one page. The longest tabulated ion corresponds to MeGlcAXyl8Ac3. 3.2. Effect of AcXEs on xylanase-released oligosaccharides Part of the hydrolysate of aspen acetyl glucuronoxylan generated with CtGH10 was heated at 100 °C to denature the xylanase. Both heated and non-heated reaction mixtures were then used as a substrate in incubations with various AcXEs. It was envisaged that in the latter reactions, xylanase degradation of oligosaccharides could proceed to a greater extent due to the deacetylation of Xylp residues. The actions of individual AcXEs are shown in Fig. 2 and summarized in Tables 1 and 2 and discussed below. Notably, as indicated in Fig. 2 but left undiscussed below, the AcXE treatments also resulted in a series of ions with masses corresponding to monoacetylated xylooligosaccharides containing one hexose. The starting mixture (Fig. 2A) did not show plausible multiple acetylated precursors for these ions. These ions are not included in Tables 1 and 2, but are marked in Fig. 2 as HexXyl2Ac (m/z 508), HexXyl3 Ac (m/z 640), HexXyl4Ac (m/z 772), HexXyl5Ac (m/z 904), HexXyl6Ac (m/z 1036), and HexXyl7Ac (m/z 1168). Their nature and origin remains to be elucidated. 3.2.1. Mode of action of ScCE1 AcXE The data in Table 1 and Fig. 2B show that ScCE1 AcXE considerably reduced the number of acetyl groups in neutral oligosaccharides. In the reaction mixtures with denatured endoxylanase all acetylated neutral xylooligosaccharides were either completely deacetylated or converted to their mono- and di-O-acetates. Deacetylated neutral oligosaccharides observed in the mixtures were as long as Xyl7. When the mixture of acetylated oligosaccharides was treated with ScCE1 AcXE in the presence of active endoxylanase, longer xylooligosaccharides and their monoacetates were further degraded and also completely deacetylated to give only

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A

B

C

D

E

Fig. 2. MALDI ToF MS of oligosaccharides generated from aspen acetyl glucuronoxylan by C. thermocellum GH10 xylanase (panel A), and analysis of their acetylation pattern after treatment with AcXEs in the presence of denatured xylanase: CE1-type AcXE from S. commune (panel B); CE5-type AcXE from T. reesei (panel C); CE4-type AcXE from S. lividans (panel D); and CE4-type AcXE from C. thermocellum (panel E). Different ions are marked by different colors (red, Xylx; green, XylxAcy; dark blue, MeGlcAXylxAcy, y N 1; light blue, MeGlcAXylxAcy, y = 1; black, other). The labeled peaks are all sodium adducts. Disodium adducts of aldouronic acids are marked by asterisks with the same color as the peak of their corresponding sodium adduct (for the sake of clarity these disodium adducts are not labeled in panel A).

three neutral non-acetylated oligosaccharides: Xyl2, Xyl3 and Xyl4 (Table 1). ScCE1 AcXE converted acidic oligosaccharides mainly to monoand di-O-acetyl derivatives. The largest detected mono-O-acetate was MeGlcAXyl8Ac (m/z 1329). Ions corresponding to completely

deacetylated MeGlcAXyl3, MeGlcAXyl4 and MeGlcAXyl5 were also observed (Fig. 2B). In the incubation mixture with active endoxylanase, the mono- and di-O-acetates of longer aldouronic acids were considerably shortened without losing the acetyl groups (Table 1). This indicated their acetylation close to or at the non-reducing end.

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Table 1 Sodium adducts of molecular ions of oligosaccharides generated from aspen acetylglucuronoxylan by CtGH10 endoxylanase and products observed after treating these oligosaccharides with AcXEs ScCE1, TrCE5 or OCE6 in the presence of active CtGH10 (“nonden.”) or denatured CtGH10 (“den.”). The TrCE5 AcXE contained contaminating endoxylanase activity, meaning that such activity was present in the reaction shown, despite the fact that CtGH10 had been denatured. See Fig. 2A, B and C for the corresponding mass spectra. Note that several of the longest MeGlcAXylxAcy products generated by GH10 alone (Fig. 2A) are not included in this table. Neutral fragments

Sodium adducts of molecular ions of neutral fragments GH10 alone

Xyl2 Xyl2Ac Xyl2Ac2 Xyl3 Xyl3Ac Xyl3Ac2 Xyl3Ac3 Xyl4 Xyl4Ac Xyl4Ac2 Xyl4Ac3 Xyl4Ac4 Xyl5 Xyl5Ac Xyl5Ac2 Xyl5Ac3 Xyl5Ac4 Xyl5Ac5 Xyl6 Xyl6Ac Xyl6Ac2 Xyl6Ac3 Xyl6Ac4 Xyl6Ac5 Xyl6Ac6 Xyl7 Xyl7Ac3 Xyl7Ac4 Xyl7Ac5 Xyl7Ac6 Xyl7Ac7

305 347 389 479 521 563

653 695 737

GH10 den. + ScCE1

GH10 nonden. + ScCE1

GH10 den. + TrCE5

305 347 389 437 479 521

305

305 347

569 611 653

701 743 785 827 869 911

437

569

Acidic fragments

437 478

569 611

569 611 653

701

701 743 785 827

833 875 917 959 1001 1043 1085 965 1091 1133 1175 1217 1259

GH10 alone

GH10 den. + OCE6

437 479

1091

Sodium adducts of molecular ions of acidic fragments

MeGlcAXyl2 MeGlcAXyl2Ac MeGlcAXyl2Ac2 MeGlcAXyl3 MeGlcAXyl3Ac MeGlcAXyl3Ac2 MeGlcAXyl3Ac3 MeGlcAXyl4 MeGlcAXyl4Ac MeGlcAXyl4Ac2 MeGlcAXyl4Ac3 MeGlcAXyl4Ac4 MeGlcAXyl5 MeGlcAXyl5Ac MeGlcAXyl5Ac2 MeGlcAXyl5Ac3 MeGlcAXyl5Ac4 MeGlcAXyl5Ac5 MeGlcAXyl6 MeGlcAXyl6Ac MeGlcAXyl6Ac2 MeGlcAXyl6Ac3 MeGlcAXyl6Ac4 MeGlcAXyl6Ac5 MeGlcAXyl6Ac6 MeGlcAXyl7 MeGlcAXyl7Ac MeGlcAXyl7Ac2 MeGlcAXyl7Ac3 MeGlcAXyl7Ac4 MeGlcAXyl7Ac5 MeGlcAXyl7Ac6 MeGlcAXyl8 MeGlcAXyl8Ac

GH10 den. + ScCE1

GH10 noden. + ScCE1

801 843 885 927

975 1017 1059 1101

GH10 den. + OCE6

495

495

495 537

495 537

627 669 711

627 669 711

627 669

627 669 711

759 801 843

759 801 843

759 801

759 801 843

891 933 975

933

537

669 711 753

GH10 den. + TrCE5

1065 1107

891 933

1065

933 975 1017

1065

1149 1191 1233 1275 1197 1239

1197

1323 1365 1407 1329

Figs. 3 and 4 show the time course of ScCE1 action on birch acetyl glucuronoxylan monitored by 1H NMR. The birch polysaccharide was used instead of aspen because it gave sharper NMR response. Signals corresponding to 2,3-di-O-acetylated Xylp residues disappeared more rapidly than the signals of 2-O- and 3-O-monoacetyled Xylp residues, showing that the enzyme has a preference for the doubly acetylated sugar, which is in accordance with a previous report [24]. The data further showed faster deacetylation of 2-O-monoacetylated xylose than of 3-O monoacetylated xylose (Fig. 4). ScCE1 was not capable of deacetylating the 3-position on Xylp residues 2-O-substituted with MeGlcA, which is a common feature of all AcXEs examined in this way [10].

3.2.3. Mode of action of OCE6 AcXE Previous studies using 1H NMR spectroscopy and polymeric substrates have shown that OCE6 efficiently deacetylates singly 2-O- and 3-O-acetylated Xylp residues and also doubly 2,3-di-O-acetylated Xylp residues in acetyl glucuronoxylan [10]. Here the action of Orpinomyces sp. AcXE was tested on the aspen acetylxylan hydrolysate after GH10 xylanase had been inactivated. All neutral and acidic xylooligosaccharides present in the starting mixture (Fig. 2A) were only partially deacetylated and persisted in the reaction mixture mainly as mono- and di-O-acetates (Table 1). The degree of acetylation was higher with shorter than longer oligosaccharides.

3.2.2. Mode of action of TrCE5 AcXE Since the TrCE5 preparation was found to be slightly contaminated with an endoxylanase, the action of this esterase on acetylated xylooligosaccharides can be considered as being evaluated in the presence of active endoxylanase despite the denaturation of the CtGH10 xylanase used to hydrolyze acetyl glucuronoxylan. Consequently, the reaction mixture obtained after treatment with TrCE5 contained relatively short xylan fragments (Fig. 2C, Table 1). After incubation with the esterase, free and monoacetylated Xyl2, Xyl3 and Xyl4 were detected, with the free oligosaccharides predominating. The complex mixture of acetylated aldouronic acids shown in Fig. 2A was converted to a mixture of short free and monoacetylated MeGlcAXylx species (x = 2–6). The mono-acetylated species were clearly more abundant than the corresponding non-acetylated species.

3.2.4. Mode of action of CE4 AcXEs The product profiles obtained for SlCE4 and CtCE4 AcXEs both showed an efficient reduction in the number of acetyl groups when added to the mixture with denatured CtGH10 xylanase (Fig. 2D, 2E and Table 2). Regardless of the polymerization degree, each neutral oligosaccharide was converted into three forms: completely deacetylated and mono- and di-O-acetylated. Only slight differences were observed between the actions of the two CE4 enzymes. Deacetylation of neutral oligosaccharides with CtCE4 (Fig. 2E) seemed somewhat less efficient than with SlCE4 (Fig. 2D), especially for longer oligosaccharides. In the reaction with CtCE4 doubly acetylated species were more abundant and for oligosaccharides larger than Xyl5 derivatives with more than three acetyl groups persisted in the reaction mixture. When the esterases were applied in the presence of active endoxylanase, the length of the neutral oligosaccharides was considerably reduced and the

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Table 2 Sodium adducts of molecular ions of oligosaccharides generated from aspen acetylglucuronoxylan by CtGH10 endoxylanase and their change after treatment with SlCE4 and CtCE4 AcXEs in the absence (GH10 den.) and in the presence (GH10 nonden.) of active GH10 endoxylanase. See Fig. 2A, D and E for the corresponding mass spectra. Note that several of the longest MeGlcAXylxAcy products generated by GH10 alone (Fig. 2A) are not included in this table. Neutral fragments

Sodium adducts of molecular ions of neutral fragments GH10 alone

Xyl2 Xyl2Ac Xyl2Ac2 Xyl3 Xyl3Ac Xyl3Ac2 Xyl3Ac3 Xyl4 Xyl4Ac Xyl4Ac2 Xyl4Ac3 Xyl4Ac4 Xyl5 Xyl5Ac Xyl5Ac2 Xyl5Ac3 Xyl5Ac4 Xyl5Ac5 Xyl6 Xyl6Ac Xyl6Ac2 Xyl6Ac3 Xyl6Ac4 Xyl6Ac5 Xyl6Ac6 Xyl7 Xyl7Ac3 Xyl7Ac4 Xyl7Ac5 Xyl7Ac6 Xyl7Ac7

305 347 389 479 521 563

653 695 737

785 827 869 911

GH10 den. + SlCE4

GH10 nonden. + SlCE4

305 347 389 437 479 521

305

569 611 653

701 743 785

833 875 917 959 1001 1043 1085 965

437 521 569

GH10 den. + CtCE4 305 347 389 437 479 521 569 611 653

Acidic fragments

GH10 nonden. + CtCE4 305

437 521 569

701 743 785 827

832 875 917 955 1001

965

1091 1133 1175 1217 1259

overall deacetylation was much more complete, limiting the product profile to free Xyl2, Xyl3 and Xyl4, and, remarkably, Xyl3Ac2. The number of acetyl groups in aldouronic acids containing three to eight Xylp residues decreased upon the SlCE4 treatment, yielding mono-acetylated and triple acetylated species as the major products (Fig. 2D, E). CtCE4 converted the acetyl derivatives of aldouronic acids to mixtures of mono-, di- and tri-O-acetates, indicating again the slight differences between the two CE4 enzymes. In the presence of an active CtGH10 xylanase, SlCE4 generated mainly monoacetates of shorter aldouronic acids such as MeGlcAXyl3 and MeGlcAXyl4, whereas longer aldouronic acids persisted as triacetates. Apparently, the acetyl groups resistant to SlCE4, which are most probably located on internal di-O-acetylated Xylp residues, prevented further hydrolysis of longer aldouronic acids by endoxylanase. Previous studies of SlCE4 action on a polymeric substrate using 1H NMR [10] have shown that the enzyme is capable of releasing acetyl groups from position 2 and position 3, but only from singly acetylated Xylp residues. This previous study also showed that SlCE4 does not deacetylate 3-O-acetyl groups on residues 2-O-substituted by MeGlcA. 1 H NMR experiments with the CtCE4 AcXE depicted in Figs. 5 and 6 gave results similar to those previously obtained for SlCE4. The enzyme deacetylated positions 2 and 3 in mono-acetylated Xylp residues at similar rates, but did not show any deacetylation of doubly acetylated Xylp residues or 3-O-acetylated residues substituted with MeGlcA.

MeGlcAXyl2 MeGlcAXyl2Ac MeGlcAXyl2Ac2 MeGlcAXyl3 MeGlcAXyl3Ac MeGlcAXyl3Ac2 MeGlcAXyl3Ac3 MeGlcAXyl4 MeGlcAXyl4Ac MeGlcAXyl4Ac2 MeGlcAXyl4Ac3 MeGlcAXyl4Ac4 MeGlcAXyl5 MeGlcAXyl5Ac MeGlcAXyl5Ac2 MeGlcAXyl5Ac3 MeGlcAXyl5Ac4 MeGlcAXyl5Ac5 MeGlcAXyl6 MeGlcAXyl6Ac MeGlcAXyl6Ac2 MeGlcAXyl6Ac3 MeGlcAXyl6Ac4 MeGlcAXyl6Ac5 MeGlcAXyl6Ac6 MeGlcAXyl7 MeGlcAXyl7Ac MeGlcAXyl7Ac2 MeGlcAXyl7Ac3 MeGlcAXyl7Ac4 MeGlcAXyl7Ac5 MeGlcAXyl7Ac6 MeGlcAXyl8 MeGlcAXyl8Ac MeGlcAXyl8Ac2 MeGlcAXyl8Ac3

Sodium adducts of molecular ions of acidic fragments GH10 alone

GH10 den. + SlCE4

537

537

669 711 753

669 711 753

801 843 885 927

801

GH10 nonden. + SlCE4

627 669

801

885

1017

1149

1197 1281

627 669

669

759 801

801 843 885

975 1017

933 975 1017

975 1017

1149

1065 1107 1149

1149

1065 1149 1191 1233 1275

GH10 nonden. + CtCE4

753

933 975 1017 1059 1101

GH10 den. + CtCE4

1197 1239 1281

1323 1365 1407 1329 1413

1329 1371 1413

4. Discussion 4.1. Mode of action of the CtGH10 xylanase on acetylated xylan The mode of action of AcXEs in this work was investigated on oligosaccharides generated from acetyl glucuronoxylan extracted from aspen by a GH10 endoxylanase from C. thermocellum (CtGH10). The pH optimum of this bacterial enzyme (pH 6.3 [16]) allowed applying the esterases, which have similar pH optima, without additional pH adjustment. The products generated by the CtGH10 xylanase alone comprised a large collection of neutral and acidic xylooligosaccharides showing high diversity in length and degree of acetylation (Fig. 2A). This shows that some of the acetyl groups prevent efficient hydrolysis of the naturally acetylated polysaccharide by CtGH10. The products of GH10 xylanase hydrolysis of alkali extracted (i.e. deacetylated) hardwood xylans are usually Xyl, Xyl2, Xyl3 and aldotetraouronic acid MeGlcA3Xyl3 [25,26]. Accumulation of MeGlcA3Xyl3 shows that the enzyme is capable of productive binding to a non-acetylated MeGlcA-substituted xylose in subsites −III, +I, and +IV (enzyme subsites are numbered here by Roman numerals to distinguish them from the numbers marking the substituted positions on Xylp residues; subsites with a plus (+) sign bind the reducing end of the substrate; subsites with a minus (−) sign bind the non-reducing end of the substrate [27,28]). Clearly (Fig. 2A), acetylation interferes with this cleavage pattern. Studies of

P. Biely et al. / Biochimica et Biophysica Acta 1830 (2013) 5075–5086

B

Free acetate

A

5081

X

X

Xyl-3Ac-2MeGlcA

1h

Xyl-2,3-Ac

Xyl, H1

Xyl-3Ac, H1

Xyl-2Ac, H1,H2 HDO

Xyl-3Ac, H3

MeGlcA, H1

Xyl-2,3-Ac, H3

Xyl-3Ac-2MeGlcA, H3

1h

Xyl-2Ac Xyl-3Ac

5h

5h

0h

0h 5.3

5.2

5.1

5.0

4.9

4.8

4.7

4.6

4.5

4.4 ppm 2.30

2.25

2.20

2.15

2.10

2.05

2.00

1.95 ppm

Fig. 3. Monitoring the action of Schizophyllum commune CE1 AcXE on birch acetyl glucuronoxylan by 1H NMR spectroscopy. Panel A shows the anomeric region, whereas panel B shows the methyl protons of the acetyl groups. The incubation time in hours is indicated. The most rapidly decreasing signals are marked with thick arrows and more slowly decreasing signals with thin arrows. Proton signals not influenced by the enzyme are marked with crossed arrows. The following designations are used: Xyl, non-acetylated Xylp; Xyl-3Ac, 3-O-acetylated Xylp; Xyl-2Ac, 2-O-acetylated Xylp; Xyl-2,3Ac, 2,3-di-O-acetylated Xylp; and Xyl-3Ac-2MeGlcA, 3-O-acetylated Xylp 2-O-linked with MeGlcA. For further quantitative analysis of the time curves, see Fig. 4.

the crystal structures of GH10 xylanases complexed with aldouronic acids and linear xylooligosaccharides have shown that the O2 groups of xyloses bound in subsites −III, +I, +III and +IV are solvent exposed [29,30], which is compatible with experimentally observed product formation from non-acetylated polysaccharide. Since the O3 groups of these sugars are equally exposed (as observed in PDB ID: 1UQY; Ref. [29]) these subsites do not seem to have steric barriers for binding xylosyl units substituted with 2-O-acetyl-, 3-O-acetyl- and/or MeGlcA-groups. Taken together, experimental and structural data suggest that the effect

16

Relative signal intensity

14 12 10 8 6 4 2

1

2

3

4

of acetylation on GH10 susceptibility seems to be governed by the effects of acetylation on xylose binding to subsites −II, −I and +II. Fig. 7 provides a summary showing how this situation would direct the cleavage of β-1,4-xylosidic linkages in deacetylated and acetylated glucuronoxylans. The main chain of deacetylated glucuronoxylan can be cleaved when Xylp carrying MeGlcA is accommodated in subsites −III, +I, +III and +IV. If the Xylp carrying MeGlcA is accommodated in subsite −III, the glycosidic linkage cleavage leads to the reducing end found later in the aldotetraouronic acid MeGlcA3Xyl3. In the complex in which the substituted Xylp would be accommodated in subsite +III, cleavage would occur at the third linkage from the substituted residue towards the non-reducing end. This could lead to generation of aldohexaouronic acid MeGlcA3Xyl5, which is cleaved to Xyl2 and aldotetraouronic acid MeGlcA3Xyl3, the shortest acidic oligosaccharide produced by GH10 xylanases [25]. The lower panel of Fig. 7 illustrates the situation for acetylated glucuronoxylan, assuming that acetylation is accepted only in the subsites that accept MeGlcA substituents (Fig. 7). However, the observation of long non-substituted oligosaccharides with high degrees of acetylation observed in the acetyl glucuronoxylan hydrolysate suggests that some of the acetyl groups must be accommodated in subsites that are restricted to MeGlcA substituents, i.e. at subsites −II, −I and +II. Clearly, there must be certain arrangements of singly and doubly acetylated Xylp residues in the xylan chain that prevent degradation of acetylated xylooligosaccharides. Disclosure of the rules that govern the enzymatic cleavage of acetylated hardwood xylan, the natural substrate of xylanases, is a challenge that requires a separate study.

5

Time (h) Fig. 4. Time resolved 1H NMR analysis of deacetylation of birch acetyl glucuronoxylan with S. commune CE1 AcXE. The graph shows internal non-acetylated Xylp residues, as determined from the anomeric region of the spectrum (see Fig. 3A) and signals of the methyl protons of acetyl groups (see Fig. 3B). Symbols: □, H-1 signal of internal non-acetylated Xylp residues; ●, 3-O-acetyl; ○, 2-O-acetyl; ▲, 2,3-di-O-acetyl, △, 3-O-acetyl-2-O-MeGlcA.

4.2. Deacetylation of xylan fragments by AcXEs The modes of action of AcXEs from four different CE families were investigated by MALDI ToF MS analysis of products generated from a mixture of acetylated oligosaccharides. The data was complemented with the data on positional specificity on polymeric substrates that were

P. Biely et al. / Biochimica et Biophysica Acta 1830 (2013) 5075–5086

A

B X

5.1

5.0

Xyl, H1

Xyl-3Ac, H1

Xyl-3Ac-2MeGlcA

HDO

Xyl-3Ac, H3

MeGlcA, H1 5.2

Xyl-2Ac, H1,H2

1h Xyl-3Ac-2MeGlcA, H3

1h

Xyl-2,3-Ac, H3

5h

Xyl-2,3-Ac

X

5h

0h

X

Xyl-2Ac Xyl-3Ac

X

5.3

Free acetate

5082

0h

4.9

4.8

4.7

4.6

4.5

4.4 ppm

2.30

2.25

2.20 2.15 2.10

2.05

2.00

1.95 ppm

Fig. 5. Monitoring of the action of Clostridium thermocellum CE4 AcXE on birch acetyl glucuronoxylan by 1H NMR spectroscopy. Panel A shows the anomeric region, whereas panel B shows the methyl protons of the acetyl groups. The incubation time in hours is indicated. Signals of protons influenced by the enzyme are marked with arrows whereas non-affected protons with crossed arrows. The following designations are used: Xyl, non-acetylated Xylp; Xyl-3Ac, 3-O-acetylated Xylp; Xyl-2Ac, 2-O-acetylated Xylp; Xyl-2,3Ac, 2,3-di-O-acetylated Xylp; and Xyl-3Ac-2MeGlcA, 3-O-acetylated Xylp 2-O-linked with MeGlcA. For further quantitative analysis of the time curves, see Fig. 6.

obtained by 1H NMR spectrometry, in a previous study (SlCE4 and OCE6) [10] and in this work (ScCE1 and CtCE4). The AcXEs used in this work utilize two different mechanisms of xylan or xylooligosaccharide deacetylation. AcXEs ScCE1, TrCE5 and OCE6 are serine type esterases [4,8] having the catalytic triad serine– histidine–aspartic acid, whereas the CE4 AcXEs from S. lividans and C. thermocellum are aspartate metalloenzymes [18,31,32]. The major difference in the mode of action between the serine-type AcXEs and CE4 AcXEs is the requirement of the CE4 esterases for a free vicinal hydroxyl group for deacetylation of position 2 or 3 [13,18,33]. This is the reason why the CE4 esterases in contrast to ScCE1 and OCE6 AcXE do not attack, 35

Relative signal intensity

30 25 20 15 10 5

1

2

3

4

5

Time (h) Fig. 6. Time resolved 1H NMR analysis of the deacetylation of birch acetylglucuronoxylan with C. thermocellum CE4. The graph shows internal non-acetylated Xylp residues, as determined from the anomeric region of the spectrum (see Fig. 5A) and signals of the methyl protons of acetyl groups (see Fig. 5B). Symbols: □, H-1 signal of internal non-acetylated Xylp residues; ●, 3-O-acetyl; ○, 2-O-acetyl; ▲, 2,3-di-O-acetyl, △, 3-O-acetyl-2-O-MeGlcA.

or attack at extremely low rate, internal doubly substituted Xylp residues. This was indeed observed; doubly acetylated neutral products mainly accumulate in the case of the CE4 enzymes (Tables 1 and 2). Based on behavior on artificial substrates [12,14] and on the results presented here, TrCE5 also appears to have the ability to deacetylate doubly acetylated Xylp residues. Table 3 summarizes the available data for the various AcXEs. Generally, the positional specificities of AcXEs derived from 1H NMR studies on polymeric substrates are in good accordance with experimentally observed product formation from acetyl glucuronoxylan oligosaccharides. In the case of serine type AcXEs, the deacetylation of neutral xylooligosaccharides ended at the stage of monoacetates or at the stage of completely deacetylated xylooligosaccharides. Some diacetates are seen in the case of ScCE1 and OCE6. The CE4 AcXEs deacetylated neutral xylooligosaccharides completely or to mono- and di-O-acetates. Acetylated acidic xylooligosaccharides, i.e. aldouronic acids, were deacetylated mainly to mono- and di-O-acetylated derivatives by ScCE1 and OCE6. All three serine-type esterases produced besides monoacetates also small amounts of completely deacetylated aldouronic acids. The products observed in the mixture treated with OCE6 suggested that this enzyme prefers as substrates longer than shorter oligosaccharides. The deacetylation of aldouronic acids with CE4 AcXEs ceased at the level of mono- and tri-O-acetyl derivatives. In the presence of CtCE4 also di-O-acetates were observed. The difference in mode of action of serine type AcXEs and aspartate metalloesterase type CEs can again be ascribed mainly to inability of the latter enzymes to attack doubly acetylated Xylp residues [10,13]. The doubly acetylated Xylp residues can be expected to remain resistant to CE4 esterases particularly when present as internal residues. The presence of xylanases during the AcXE treatments led to a clear reduction in fragment size and to increased deacetylation. This clearly shows that the xylanases are hindered by acetyl groups, whereas the deacetylases are hindered by acetylations and/or substitutions on adjacent sugars which are removed by xylanase cleavage. Notably, the data in Tables 1 and 2 may give the impression that treatment with AcXEs alone leads to some depolymerization, since longer products disappear

P. Biely et al. / Biochimica et Biophysica Acta 1830 (2013) 5075–5086

A

-III

-II

-I

+I

+II

+III

5083

+IV

-Xyl- Xyl -Xyl- Xyl - Xyl- Xyl - Xyl - Xyl MeGlcA

B

+I

MeGlcA

-III,+III

MeGlcA MeGlcA

+I

-III

+III

+I

-III,+IV

+III

+I

Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-XylMeGlcA

MeGlcA +I

C 3Ac 3Ac

3Ac 3Ac

3Ac

MeGlcA

3Ac

MeGlcA

3Ac

3Ac

3Ac

Xyl -Xyl-Xyl-Xyl- Xyl -Xyl-Xyl-Xyl-Xyl-Xyl-Xyl- Xyl -Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl2Ac

2Ac MeGlcA

MeGlcA

2Ac 2Ac

MeGlcA

2Ac

MeGlcA

Fig. 7. Hydrolysis of glycosidic linkages in glucuronoxylan by GH10 xylanases. Panel A shows the substrate binding subsites of the CtGH10 xylanase depicted by indentations numbered from −III to +IV. The large indentations correspond to subsites capable of accommodation of Xylp residues substituted with MeGlcA. Panel B illustrates the expected cleavage sites (arrows) in a hypothetical deacetylated glucuronoxylan; the numbers over the cleavage sites indicate which of the enzyme's subsites (−III to +IV) accommodated Xylp residues substituted with MeGlcA. Panel C illustrates how acetylation could reduce cleavability and is based on the assumption that acetylation in subsites −I and +II is incompatible with productive binding. See text for further explanation.

even in the absence of active xylanase. This, however, is due to a decrease in solubility upon deacetylation, which lowers ionization efficiency. Putative locations of acetyl groups resistant to AcXEs can be proposed on the basis of the following data: i) positional specificities analyzed by NMR (see Table 3); ii) AcXE activities on acetylated artificial substrates [4,11–14]; iii) known requirement of CE4 AcXEs for a free vicinal OH group [13,18,33], iv) inability of AcXEs to hydrolyze a 3-Oacetylated Xylp residue with a MeGlcA-substituent at vicinal O2, and v) possibility of acetyl group migration along the Xylp ring [34,35,21]. The resistant monoacetates of neutral xylooligosaccharides correspond most probably to products with a 4-O-acetylated Xylp residue at the non-reducing end, which results from the spontaneous migration of the acetyl group from position 3 (Fig. 8A). The di-O-acetates resistant to CE4 AcXEs correspond to compounds containing one 2,3-di-Oacetylated internal Xylp residue (Fig. 8B and C). We assume that 2,3-diO-acetylated Xylp residues at the non-reducing end of oligosaccharides

would slowly disappear even with CE4 AcXEs due to their spontaneous change into 2,4- or 3,4-di-O-acetyl derivatives as a result of the acetyl group migration [34,35,21]. It is unlikely that resistant acetyl groups occur at the reducing end of the oligosaccharides because subsite −I of the substrate binding site of GH10 xylanases [29,30] should not allow binding of a Xylp residue substituted at positions 2 and/or 3. Notably, in a recent study on C. lucknowense AcXEs of family CE1 and CE5, it was suggested that these enzymes produce neutral xylooligosaccharides monoacetylated close to the non-reducing end or at position 4 at the non-reducing end [9]. All AcXEs also reduced the number of acetyl groups in acidic xylan fragments considerably. The serine-type AcXEs deacetylated aldouronic acids mainly to mono- and di-O-acetates. The aspartatemetalloesterase-type AcXEs yielded aldouronic acids with one or three acetyl groups, and, in the case of CtCE4, also diacetates. In this case an additional resistance mechanism is obvious, namely the presence of 3-O-acetyl groups on the Xylp residue α-1,2-substituted with MeGlcA

Table 3 Positional specificity of AcXEs on acetyl glucuronoxylan and their action on neutral and acidic xylooligosaccharides generated from the polysaccharide by CtGH10 endoxylanase. Major acidic xylooligosacch. formed after AcXEs treatment (GH10 denatured)

Major neutral xylooligosacch. formed after AcXE treatment (GH10 denatured)

Major neutral xylooligosacch. formed after AcXEs treatment (GH10 active)

ScCE1 2,3-di-O-Ac Xylp, 2- and 3-Omonoacetylated Xylp

Deacetylated and mono- and di-O-acetates

TrCE5 Not tested

Deacetylated and monoacetylated Xyl2, Xyl3 and Xyl4; Xyl5 completely deacetylated Short oligosaccharides deacetylated and mono- and di-O-acetylated; longer oligosaccharides as monoacetates Deacetylated and mono- and di-O-acetyl derivatives

Deacetylated Xyl2, Xyl3 and Xyl4 Short aldouronic acids deacetylated and mono- and di-O-acetylated; mono- and di-O-acetyl derivatives of longer aldouronic acids b Deacetylated and mono- acetylated shorter aldouronic acids

AcXE

OCE6

Deacetylated Xylp residues in polysaccharide determined by 1 H NMR a

2,3-di-O-Ac Xylp, 2- and 3-Omonoacetylated Xylp

SlCE4 2- and 3-O-monoacetylated Xylp residues CtCE4 2- and 3-O-monoacetylated Xylp residues

a b

Deacetylated and mono- and di-O-acetyl derivatives; some species tri- or tetraacetylated

Major acidic xylooligosacch. formed after AcXEs (GH10 active) Short aldouronic acids, deacetylated and mono- and di-O-acetylated b

Not tested

Short aldouronic acids deacetylated and mono- and di-O-acetylated; longer aldouronic acids as monoacetates

Not tested

Deacetylated Xyl2 and Xyl3; Xyl3 also as di-O-acetate

Mono- and tri-O-acetylated aldouronic acids

Deacetylated Xyl2 and Xyl3; Xyl3 also as di-O-acetate

Mono- and tri-O-acetylated aldouronic acids

Short mono- and tri-Oacetylated; MeGlcAXyl3 partially deacetylated Short aldouronic acids, deacetylated and monoacetylated; longer also as di- and tri-O-acetylates

None of the AcXE is capable of deacetylating O3 next to a MeGlcA-substituted O2. Note that the TrCE5 preparation was contaminated with endoxylanase activity; see text for more details.

5084

P. Biely et al. / Biochimica et Biophysica Acta 1830 (2013) 5075–5086

A

O

O

O

O HO

OH

CH3

B

O HO

O O

OH HO

OH

n O

O HO HO

O HO

OH

O

C

O O

OH

O HO

OH

CH3

OH HO

O CH3

OH n≥2

O

O

O O HO

O

O O

O

OH

CH3 O

O

O

O O

OH O

CH3

OH HO

O

OH n≥2

O

CH3

Fig. 8. Possible structural features of acetylated neutral oligosaccharides that resist deacetylation with the AcXEs tested in this study. The arrows indicate migration of the acetyl group from O3 to O4. Note that resistance to deacetylation varies among the AcXEs. For example, the doubly acetylated internal xyloses are only resistant to deacetylation by CE4-type AcXEs. See text for more details.

A

O

O HO O H3C

O O

O HO

O O

OH HO

OH

OH n

OH

HO H3CO

O COOH

B O

O

O

O HO

H3C

OH

O HO

O

O

O

O HO

OH

OH HO

O

OH

OH

n≥2

HO H3CO

O COOH

C O

O

O

O HO

H3C

OH

O HO

O

O

O

O O

OH H3C

O

OH HO

O

OH n≥2

OH

HO H3CO

O COOH

D O

O HO HO

O

O HO

O

OH

O O

OH H3C

HO H3CO

O O

O

OH HO

O

OH n≥2

H3C

O

O COOH

E O

O

O H3C

O HO

O OH

HO H3CO

O HO

O

O

O

O O

OH H3C

O O

H3C

OH HO

OH n≥2

O

O COOH

Fig. 9. Possible structural features of acetylated aldouronic acids that resist deacetylation with the AcXEs tested in this study. The arrows indicate migration of the acetyl group from O3 to O4. In the case of a non-substituted non-reducing end (red arrows), migration is a prerequisite for resistance. In the case of a MeGlcA-substituted reducing end (black arrows), both the O3 and the O4 acetylations could be resistant.

P. Biely et al. / Biochimica et Biophysica Acta 1830 (2013) 5075–5086

(Fig. 9). Although, GH10 xylanases can accommodate such substituted Xylp residues in subsites −III, +I, +III and +IV [29,30], such Xylp residue can be expected to be located mainly at the non-reducing end (Fig. 9A, E), for the same reasons as discussed above (Fig. 7). Other types of monoacetates could have the acetyl group at position 4 of the non-reducing end or a 3-O-acetylated MeGlcA-substituted Xylp as the third residue from the non-reducing end (which during cleavage was accommodated in subsite +III; Fig. 9B). Diacetates could contain one acetyl group at O4 on the non-reducing end and a 3-O-acetyl group on an internal MeGlcA-substituted Xylp residue (Fig. 9C). Other diacetates could contain one internal doubly acetylated Xylp residue plus a single residue substituted with MeGlcA but not acetylated (Fig. 9D). The triacetates observed with CE4 AcXEs presumably contain both the 3-O-acetylated Xylp residue substituted with MeGlcA at the non-reducing end and a doubly acetylated internal Xylp residue (Fig. 9E). 5. Conclusions This work extends current knowledge on microbial degradation of native acetylated plant xylans. Our data clearly show that AcXEs of all four tested CE families are able to deacetylate almost all positions in hardwood acetylglucuronoxylan. An exception is the 3-O-acetyl group on Xylp residues α-1,2-substituted substituted with MeGlcA. Removal of this group in the polymer or in acetylated oligosaccharides (where the group may migrate to O4) requires an additional deacetylase. The MeGlcA group apparently functions as a steric barrier of deacetylation of position 3 for all AcXEs, including those that do not require free vicinal OH-groups to complete deacetylation of position 2 or 3. One candidate for deacetylation of O4 at the non-reducing end of neutral xylooligosaccharides and/or the 3- or 4-positions at the nonreducing end Xylp residues substituted MeGlcA (Figs. 8 and 9) is the T. reesei CE16 acetyl esterase, an exo-acting deacetylase, a component of the secretome of the fungus produced on cellulose [4,19,36,37]. This enzyme shows preference for deacetylation of positions 3 and 4 on 4-nitrophenyl β-D-xyloside [36,14] and catalyzes transacetylation to position 3 of the non-reducing residues of glycosides or oligosaccharides of pentoses and hexoses [4,38,39]. The 3-O-acetylated products were selectively formed only in the case of hexose oligosaccharides such as cellooligosaccharides. In xylooligosaccharides the 3-O-acetyl group migrated to neighboring positions, so a mixture of monoacetyl derivatives was formed [39]. This aspect of hardwood acetyl glucuronoxylan degradation is undoubtedly important for achieving complete deacetylation and saccharification, and is under current investigation. An AcXE liberating the 3-O-acetyl group on internal Xylp residues substituted with MeGlcA awaits its discovery. Acknowledgements The authors thank Drs. Claude Dupont and Dieter Kluepfel (Institute of Armand Frappier, Laval, Canada) and Profs. Carlos M.G.A. Fontes (Universidade Técnica de Lisboa, Portugal), Gideon J. Davies (University of York, UK) and Maija Tenkanen (University of Helsinki, Finland) for generously supplying the acetylxylan esterases. This work was supported by grants from the Slovak Academy of Sciences grant agency VEGA 2/ 0001/10 and VEGA 2/0116/10, by the Research and Development Operational Programme ITMS 26220120054 funded by the ERDF, by grant 214613 from the Norwegian Research Council, and by the FP7 project Waste2Go under contract 308363 with the European Commission. References [1] H.O. Bouveng, P.J. Garegg, B. Lindberg, Position of O-acetyl groups in birch xylan, Acta Chem. Scand. 14 (1960) 742–748. [2] T.E. Timmel, Recent progress in the chemistry of wood hemicelluloses, Wood Sci. Technol. 1 (1967) 45–70. [3] K.C.B. Wilkie, Hemicellulose, Chem. Tech. 13 (1983) 306–319.

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