Biochimica et Biophysica Acta 1622 (2003) 82–88 www.bba-direct.com

Mode of action of acetylxylan from Streptomyces lividans: a study with deoxy and deoxy-fluoro analogues of acetylated methyl h-D-xylopyranoside

Peter Bielya,*,Ma´ria Mastihubova´ a, Gregory L. Coˆte´ b, Richard V. Greeneb

a Institute of Chemistry, Slovak Academy of Sciences, Dubravska cesta 9, 84238 Bratislava, Slovak Republic b Fermentation Biotechnology Research Unit, National Center for Agricultural Research, Agricultural Research Service, United States Department of Agriculture, Peoria, IL, USA Received 6 March 2003; received in revised form 26 May 2003; accepted 12 June 2003

Abstract

Streptomyces lividans acetylxylan esterase removes the 2- or 3-O-acetyl groups from methyl 2,4-di-O-acetyl- and 3,4-di-O-acetyl h-D- xylopyranoside. When the free hydroxyl group was replaced with a hydrogen or fluorine, the rate of deacetylation was markedly reduced, but regioselectivity was not affected. The regioselectivity of deacetylation was found to be independent of the prevailing conformation of the substrates in solution as determined by 1H-NMR spectroscopy. These observations confirm the importance of the vicinal hydroxyl group and are consistent with our earlier hypothesis that the deacetylation of positions 2 and 3 may involve a common ortho-ester intermediate. Another possible role of the free vicinal hydroxyl group could be the activation of the acyl leaving group in the deacetylation mechanism. Involvement of the free hydroxyl group in the –substrate binding is not supported by the results of inhibition experiments in which methyl 2,4-di-O-acetyl h-D-xylopyranoside was used as substrate and its analogues or methyl h-D-xylopyranoside as inhibitors. The enzyme requires for its efficient action the trans arrangement of the free and acetylated hydroxyl groups at positions 2 and 3. D 2003 Elsevier B.V. All rights reserved.

Keywords: Acetylxylan esterase; Streptomyces lividans; Deacetylation of carbohydrate

1. Introduction The observed regioselectivity of deacetylation of low-mo- lecular mass substrates, deacetylation of positions 2 and 3, Acetylxylan (AcXEs) are components of micro- was consistent with the function of the in acetyl- bial xylanolytic systems that liberate acetic acid from par- xylan degradation [5,6,8]. One of the investigated enzymes, tially acetylated 4-O-methyl-D-glucurono-D-xylan, the main AcXE from Streptomyces lividans, a member of the carbo- hardwood hemicellulose, or from its fragments, acetylated hydrate esterase family 4 [http://afmb.cnrs-mrs.fr/CAZY/ xylooligosaccharides, generated by the action of endo-h-1,4- index.html], showed unique behaviour [6]. It attacked very xylanases [1–4]. Studies of substrate specificity of several slowly fully acetylated h-D-xylopyranoside, but the first AcXEs pointed to the ability of these enzymes to operate deacetylation of the compound was immediately followed effectively on a variety of acetylated carbohydrates, includ- by deacetylation at the neighbouring position so that a ing acetylated monosaccharides and their glycosides [5–7]. double deacetylation at positions 2 and 3 took place. The reason for the double deacetylation of methyl 2,3,4-tri-O- h Abbreviations: AcXE, acetylxylan esterase (EC 3.1.1.72); 2,3,4-tri-O- acetyl -D-xylopyranoside became better understood after Ac-Me-h-Xylp, methyl 2,3,4-tri-O-acetyl-h-D-xylopyranoside; 2,4-di-O- finding that two of the diacetates, 2,4-di-O-acetyl and 3,4-di- Ac-Me-h-Xylp, methyl 2,4-di-O-acetyl-h-D-xylopyranoside; 2-de-3,4- O-acetyl methyl h-D-xylopyranoside, were deacetylated at a di-O-Ac-Me-h-Xylp, methyl 2-deoxy-3,4-di-O-acetyl-h-D-xylopyranoside; rate several orders of magnitude faster than methyl 2,3,4-tri- 2-de-2-F-3,4-di-O-Ac-Me-h-Xylp, methyl 2-deoxy-2-fluoro-3,4-di-O-ace- O-acetyl h-D-xylopyranoside and methyl 2,3-di-O-acetyl h- tyl-h-D-xylopyranoside; Me-h-Xylp, methyl h-D-xylopyranoside * Corresponding author. Tel.: +42-12-5941-0275; fax: +42-12-5941- D-xylopyranoside. The enzyme deacetylates position 2 or 3 0222. efficiently if the adjacent OH-group is not esterified [6]. E-mail address: [email protected] (P. Biely). Since it was difficult to imagine that the enzyme has an equal

0304-4165/03/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0304-4165(03)00130-2 P. Biely et al. / Biochimica et Biophysica Acta 1622 (2003) 82–88 83 ability to deacetylate positions 2 and 3 under these circum- enzyme belongs to family 4 of carbohydrate esterases [http:// stances, we hypothesized that the deacetylation of position 2 afmb.cnrs-mrs.fr/~pedro/CAZY/index.html]. or 3 involves the same intermediate, a five-membered transition state, an ortho ester, which is involved in the 2.2. Acetylated carbohydrate substrates acetyl group migration along the pyranoid sugar rings [5,9]. Similar ortho-amino acid intermediates of amino acid– Fully acetylated methyl h-D-xylopyranoside (2,3,4-tri-O- tRNA were proposed as transition states in the reaction of Ac-Me-h-Xylp) was obtained by of commercially peptidyltransferases (aminoacyl-tRNA synthases) [10]. available methyl h-D-xylopyranoside (Sigma) with acetic Strong support for such a mechanism was obtained in acid anhydride in pyridine. Methyl 2,4-di-O-acetyl-h-D-xylo- experiments with 2V-deoxy- and 3V-deoxyaminoacyl deriva- pyranoside (2,4-di-O-Ac-Me-h-Xylp) was supplied by Drs. tives used as amino acid donors and acceptors [11]. Pavol Kova´e` and Ja´n Hirsch (Institute of Chemistry, SAS, The possibility of the existence of a five-membered Bratislava) and methyl 3,4-di-O-acetyl-h-D-xylopyranoside intermediate in the mechanism of deacetylation of positions (3,4-di-O-Ac-Me-h-Xylp) was prepared by enzymatic acet- 2 and 3 by AcXEs was recently weakened after resolution of ylation of methyl h-D-xylopyranoside with PS (Ama- the three-dimensional structures of AcXEs from Penicillium no) as described by Lo´pez et al. [17]. 2-Deoxy-, 3-deoxy-, 2- purpurogenum (AXEII) [12] and Trichoderma reesei [13]. deoxy-2-fluoro- and 3-deoxy-3-fluoro derivatives of methyl Both enzymes belong to a different carbohydrate esterase h-D-xylopyranoside diacetates (2-de-3,4-di-O-Ac-Me-h- family than the S. lividans AcXE. The structure of T. reesei Xylp, 2-de-2-F-3,4-di-O-Ac-Me-h-Xylp, 3-de-2,4-di-O-Ac- AcXE [13] does not lend itself to our hypothesis, and as an Me-h-Xylp and 3-de-3-F-2,4-di-O-Ac-Me-h-Xylp)were explanation of the high rate of deacetylation of positions 2 prepared by a new route via 2,3-anhydropentosides [14]. and 3 in methyl h-D-xylopyranoside diacetates, the authors propose a 180j reoriented binding of 2,4-di-O-acetylated or 2.3. Enzymatic deacetylation 3,4-di-O-acetylated substrates. Although AcXE from S. lividans differs from T. reesei AcXE in many aspects, for Reactions were performed in 10 mM homogeneous example, it does not seem to be a serine-type esterase (C. solutions of acetylated derivatives in 0.1 M sodium phos- Dupont and V. Puchart, private communication), the idea that phate buffer, pH 6.0, at 40 jC. S. lividans AcXE concen- low-molecular mass substrates can complex with the enzyme tration varied with the substrates. Solutions of 2,4-di-O-Ac- in different orientations should also be considered in the case Me-h-Xylp and 3,4-di-O-Ac-Me-h-Xylp, which are the of S. lividans AcXE. substrates in which spontaneous acetyl group migration To get more insight into the mechanism of action of S. occurs in aqueous media, were always freshly prepared in lividans AcXE and to obtain more information relevant to the the shortest possible time before enzyme addition and their formation of the hypothesized five-membered ortho-ester enzymatic treatment was never longer than 15–20 min. intermediate, the performance of the enzyme on 2,4- and 3,4- Aliquots of the reaction mixtures were taken to analyze the diacetates and 2,3,4-triacetate of methyl h-D-xylopyranoside products and calculate the rate of the first deacetylation. was compared with its action on some recently synthesized diacetates of 2-deoxy-, 3-deoxy-, 2-deoxy-2-fluoro- and 3- 2.4. Product analysis deoxy-3-fluoro-analogues of methyl h-D-xylopyranoside [14]. Structures of these four analogues exclude the possi- Reaction mixtures of all substrates were analyzed by TLC bility of acetyl group migration in the molecules before or in on glass plates of Silica gel G-60 (Merck) in ethyl acetate– the course of the first enzymatic deacetylation, which means benzene–2-propanol (2:1:0.1, v/v). Due to different sensi- that the formation of the five-membered intermediate is not tivities of detection, the derivatives were divided into two possible. Thus, the role of the OH-groups adjacent to the groups. The first group contained 2,3,4-tri-O-Ac-Me-h- acetyl groups in enzyme–substrate interactions may be Xylp,2,4-di-O-Ac-Me-h-Xylp,3,4-di-O-Ac-Me-h-Xylp, evaluated. 2-de-2-F-3,4-di-O-Ac-Me-h-Xylp, and 3-de-3-F-2,4-di-O- Ac-Me-h-Xylp. One-microliter aliquots of the reaction mix- tures of these compounds, corresponding to 10 nmol of the 2. Materials and methods starting substrate, were chromatographed and compounds detected on dried chromatograms with N-(1-naphtyl)ethyle- 2.1. Enzyme nediamine dihydrochloride reagent [18] modified so that it could be used by pouring on the plates. The modified reagent AcXE used in this work was a product of a genetically was prepared by dissolving 1.8 g of the reagent in a mixture modified strain of S. lividans IAF43, which overproduces the of 200 ml of ethanol and 20 ml of concentrated H2SO4. esterase together with xylanase B [15]. The enzyme purified Deoxy analogues showed much higher sensitivity of from the culture fluid as described [16] was a generous gift detection with orcinol reagent than with the N-(1-naphtyl)e- from Dr. Claude Dupont (Institute Armand Frappier, Uni- thylenediamine dihydrochloride reagent. Aliquots of the versite´ du Que´bec, Ville de Laval, Que´bec, Canada). The reaction mixtures containing 2-de-3,4-di-O-Ac-Me-h-Xylp, 84 P. Biely et al. / Biochimica et Biophysica Acta 1622 (2003) 82–88

3-de-2,4-di-O-Ac-Me-h-Xylp applied on Silica gel were 2 or 2.5. Substrate conformations 4 Al in volume, corresponding to 20 and 40 nmol of the starting substrates. The compounds were visualized on dried The preferred conformations in aqueous solutions (C-1 or chromatograms by pouring orcinol reagent prepared by 1-C) of the acetylated carbohydrates used as AcXE sub- dissolving 2 g of orcinol in a mixture of 180 ml of ethanol strates were deduced from the J1,2 coupling constants deter- 1 and 20 ml of concentrated H2SO4. mined by H-NMR spectroscopy. The J1,2 in the range 6.5– The development of spots was done under standard 7.5 corresponds to the C-1 conformation, while the values in conditions in a ventilated oven at 120 jC. The developed the range 2.0–3.0 indicate a 1-C conformation [19]. chromatograms were scanned in a reflectance mode and the images were analyzed by densitometry using Un-Scan-It 2.6. Isolation and structure of products of the first software (Silk Scientific, Orem, UT, USA). This is compa- deacetylation rable to the method published by Bounias [18]. The method gave satisfactory linear relationship between Products of the first deacetylation were isolated from the amount of compounds of the first group (detected with larger scale enzyme–substrate mixtures by TLC as described N-(1-naphthyl)ethylenediamine dihydrochloride reagent) above. Reaction mixtures containing 10 mg of the starting and the square of spot density up to 10 nmol of compounds. substrate treated with AcXE for an appropriate time were In the case of deoxy derivatives detected with the orcinol chromatographed on three or four analytical TLC Silica gel reagent, the best linearity between the amount of com- G-60 plates. The desired products were localized using guide pounds and spot density was observed when the spot density strips on which the compounds were visualized as given was plotted not as value to the exponent 2 but to the above. Silica gel zones corresponding to the desired products exponent 1.5 (i.e., amount vs. [density]1.5). Multiple data were removed from the glass plates and extracted with ethyl on the rate of the first deacetylation were used to calculate acetate or anhydrous ethanol. Dried residues were dissolved 1 the standard deviation. in D2O and their structure established by H-NMR spectros-

Table 1 Action of S. lividans AcXE on acetylated derivatives of methyl h-D-xylopyranoside and its deoxy and deoxy-fluoro analogues

Specific activities of the enzyme were determined at 10 mM substrate concentration in 0.1 M sodium phosphate buffer (pH 6.0) at 40 jC. Relative rates of the 1 first deacetylation are confronted with the conformation of the substrates assessed on the basis of the coupling constant J1,2 obtained from the H-NMR spectra 1 1 1 in D2O. Position of the acetyl group in the products of the first deacetylation was obtained by H-NMR spectroscopy from H– H COSY spectra. *The substrate in which the first slow deacetylation is immediately followed by the second fast deacetylation. **Determined from initial rates of the first deacetylation (with exception of 2,3,4-tri-O-Ac-Me-h-Xylp, the first deacetylation is more or less the final one). 1 ***From H-NMR spectra in D2O. P. Biely et al. / Biochimica et Biophysica Acta 1622 (2003) 82–88 85 copy. Two-dimensional 1H–1H COSY spectra [20] were sufficient to deduce the positions of the acetyl groups in the compounds.

3. Results and discussion

3.1. Role of the vicinal OH-group in the deacetylation Fig. 2. Hexagonal projections of 2,4-di-O-Ac-Me-h-Xylp and 3,4-di-O-Ac- mechanism Me-h-Xylp, and possible recognition of their substituents at positions 2 and 3 by AcXE. 3,4-di-O-Ac-Me-h-Xylp is depicted to interact with the The experimental method for measuring the rate of the enzyme in a reverse orientation. first deacetylation of acetylated methyl h-D-xylopyranosides and their analogues used in this work confirmed previous Replacement of the free hydroxyl group in 2,4-di-O-Ac- results obtained by GLC [6]. S. lividans AcXE deacetylates Me-h-Xylp and 3,4-di-O-Ac-Me-h-Xylp by hydrogen or 2,4-di-O-Ac-Me-h-Xylp and 3,4-di-O-Ac-Me-h-Xylp much fluorine affected the rate of deacetylation in a dramatic way faster than 2,3,4-tri-O-Ac-Me-h-Xylp (Table 1). The first (Table 1). The specific activity of AcXE for 2-deoxy- and 3- acetyl group in fully acetylated glycoside is removed almost deoxy analogues of methyl h-D-xylopyranoside diacetates four thousand times more slowly than in 2,4-di-O-Ac-Me-h- dropped to the range of values exhibited by the enzyme with Xylp, and almost 800 times more slowly than in 3,4-di-O- the fully acetylated methyl h-D-xylopyranoside. The specif- Ac-Me-h-Xylp. However, the second deacetylation of 2,3,4- ic activity of the enzyme with fluoro-analogues was 3–25 tri-O-Ac-Me-h-Xylp proceeded almost simultaneously since times higher than with the corresponding deoxy-analogues, 4-O-Ac-Me-h-Xylp was the only major deacetylation prod- indicating slightly better conditions for substrate deacetyla- uct. The highest rate of deacetylation of all the substrates tion in derivatives containing fluorine instead of hydrogen. tested, which was for 2,4-di-O-Ac-Me-h-Xylp,when This could be due to electron-withdrawing effect of fluorine, expressed as specific activity referred to 1 mg of AcXE deepening the electron deficiency on the acetyl group protein (Table 1), suggests that the enzyme has a much carbon. However, the rates of deacetylation of the com- higher affinity for the 2-O-acetyl group than for the 3-O- pounds containing the hydroxyl group in the position of acetyl group if the adjacent OH-group (at position 2 or 3) is fluorine were still one or two orders of magnitude greater non-acetylated. than those with fluoro derivatives. At first glance, the above observations are in accord with the hypothesis of the formation of the five-membered ortho- ester intermediate which can be formed only in those acetylated methyl h-D-xylopyranosides which contain one hydroxyl group free at positions 2 and 3 and one acetylated (Fig. 1). We would like to stress here that we have viewed the formation of the cyclic intermediate as a step preceding the generally accepted mechanism of esterases [13], which is based on the triad of catalytic amino acids. However, the formation of the intermediate could be an enzyme-facilitated step involved in the removal of the acetyl group from either of the two different positions even if S. lividans AcXE would not be a serine-type esterase (V. Puchart and C. Dupont, personal communication).

3.2. Reversed substrate binding

A recent suggestion that the high rate of enzymic deace- tylation of positions 2 and 3 by T. reesei AcXE could be a result of differently oriented binding of 2,4-di-O-Ac-Me-h- Xylp and 3,4-di-O-Ac-Me-h-Xylp [13] should also be taken into consideration in the case of the S. lividans enzyme. Fig. 2, in which the two methyl h-D-xylopyranoside diacetates are depicted also in the hexagonal projection, clearly illus- Fig. 1. Hypothesized five-membered ortho ester intermediate involved in trates that the spatial arrangement of substituents at C-2 and deacetylation of h-D-xylopyranoside positions 2 and 3 in the presence of the vicinal free hydroxyl group at positions 3 and 2. Notice two possible C-3 in 2,4-di-O-Ac-Me-h-Xylp and 3,4-di-O-Ac-Me-h-Xylp diastereoisomers of the intermediate. is identical when looking at one of the molecules from 86 P. Biely et al. / Biochimica et Biophysica Acta 1622 (2003) 82–88

function in the substrate binding as hydrogen bond accept- ors. Whether they function as hydrogen bond donors is not yet known. We have tried to obtain experimental evidence for the role of the free OH-groups at position 2 or 3 by testing the effect of underivatized Me-h-Xylp on the rate of deacetylation of 2,4-di-O-Ac-Me-h-Xylp and 3-de-2,4-di-O-Ac-Me-h-Xylp. As shown in Fig. 3A, no inhibition of the deacetylation of the first derivative (5 mM) was observed at 25 and 125 mM concentration of Me-h-Xylp, and of the 3-deoxy-analogue (5 mM) at 5 and 25 mM Me-h-Xylp (Fig. 3B). By TLC, we examined also the effect of other analogues of acetylated Me-h-Xylp. No evidence was obtained for efficient inhibi- tion of 2,4-di-O-Ac-Me-h-Xylp deacetylation. These obser- vations support the idea that the OH-group adjacent to the acetyl group at position 2 or 3 plays an important role in the reaction mechanism rather than in the binding of the sub- strate. One of the roles of the vicinal hydroxyl groups could be the activation of the ester carbonyl group by a hydrogen bond formation with the vicinal hydroxyl group (Fig. 4).

3.3. Role of the substrate conformation

Being aware that the replacement of the OH-groups on a pyranoid ring by hydrogen may lead to a conformational change [19], we examined the preferred conformation of all substrates in heavy water on the basis of J1,2 coupling constants by 1H-NMR spectroscopy. As shown in Table 1, both deoxy analogues of Me-h-Xylp diacetates show low J1,2 values, indicating that their preferred conformation is 1- Fig. 3. Effect of Me-h-Xylp on deacetylation of 5 mM 2,4-di-O-Ac-Me-h- C, in contrast to a C-1 conformation of all other substrates. Xylp (Part A) and 5 mM 3-de-2,4-di-O-Ac-Me-h-Xylp (Part B) by AcXE Regardless the preferred conformation, all substrates were from S. lividans. Symbols in Part A: control without Me-h-Xylp,(o); 25 mM Me-h-Xylp,(.); 125 mM Me-h-Xylp,(D). Symbols in Part B: converted by the enzyme to their 4-monoacetyl derivatives in control without Me-h-Xylp,(o); 5 mM Me-h-Xylp,(.); 25 mM Me-h- high yields. The 4-monoacetates of deoxy and deoxy-fluoro Xylp,(D). analogues of Me-h-Xylp are described here for the first time. The fact that the product of the first deacetylation of 3- opposite side (Fig. 2). If the enzyme is unable to differentiate deoxy-2,4-di-O-Ac-Me-h-Xylp is also its 4-monoacetate between the C-4 acetyl group and the C-1 methoxyl group, (Fig. 5), suggests that the substrate conformation does not and to direct the substrates into one type of productive influence the regioselectivity of the enzymatic deacetylation. binding, we admit that the fast deacetylation of positions 2 The enzyme apparently attacks only the ‘‘correct’’ confor- and 3 in the presence of the adjacent free hydroxyl group at mation, or the conformation may change upon binding to the positions 3 and 2 could be a result of the formation of two enzyme. The mixture of 3-monoacetyl and 4-monoacetyl types of productive complexes with methyl h-D-xylopyrano- side diacetates in which the methyl aglycon points to either of two different directions in the substrate of S. lividans AcXE. The replacement of the OH-group by hy- drogen or fluorine at position 2 or 3 could have serious consequences for the deacetylation rate if the role of the OH- group was not involved in the formation of the ortho-ester intermediate but rather in the substrate binding. Such a role of the OH-group at position 2 or 3 could be envisaged as a donor of a crucial hydrogen bond involved in the substrate binding. Fluorine, which may function as a hydrogen bond Fig. 4. Activation of the ester carbonyl group via hydrogen bonding with acceptor only, does not eliminate the loss of the enzyme the vicinal hydroxyl group in 2,4-di-O-Ac-Me-h-Xylp and 3,4-di-O-Ac- activity seen when the OH-group is replaced by hydrogen. Me-h-Xylp (the latter substrate would be bound to the enzyme in different This indirectly suggests that the crucial OH-groups do not orientations as indicated in Fig. 2). P. Biely et al. / Biochimica et Biophysica Acta 1622 (2003) 82–88 87

Fig. 5. Two-dimensional 1H–1H COSY spectrum of mono-O-acetyl derivative formed by S. lividans AcXE from 3-de-2,4-di-O-Ac-Me-h-Xylp. The assignments point to the presence of the acetyl group in position 4 (see the formula). derivatives as products of 2-deoxy-3,4-di-O-Ac-Me-h-Xylp substrates or inhibitors confirmed that the free hydroxyl is believed to be a consequence of acetyl group migration, group plays an important role in the deacetylation mecha- since the deacetylation proceeded rather slowly even with a nism rather than in the substrate binding. Since we cannot large excess of AcXE. The fact that the 4-monoacetyl exclude binding of the Me-h-Xylp diacetates to S. lividans derivative was the product of deacetylation of all tested AcXE in two different orientations, the mechanism of deace- substrates is evidence that the enzyme cannot accommodate tylation involving the five-membered ortho-ester intermedi- in its substrate-binding site the trans arrangement of the ate has not been confirmed. Therefore, our future strategy is acetyl group and hydroxyl group which occurs on carbon directed toward synthesis of diacetates of h-D-xylopyrano- atoms C-3 and C-4. This arrangement is, in relation to the sides with aglycones larger than the methyl group, so they area of the pyranoid ring, spatially reversed relative to the could direct the substrates into one type of productive arrangement of the two groups on C-2 and C-3 in 2,4-di-O- binding, corresponding to that of a xylan. Naturally, further Ac-Me-h-Xylp and 3,4-di-O-Ac-Me-h-Xylp. In this connec- progress in understanding the action mechanism of S. lividans tion, it would be interesting to examine the action of S. AcXE may be expected once its three-dimensional structure lividans AcXE on acetyl derivatives of L-lyxose, in which the is elucidated. It will be of interest to find out whether the spatial arrangement of groups at C-3 and C-4 match the C-2 presence of the free vicinal hydroxyl group is also important and C-3 arrangement in D-xylopyranosides. for the action of other carbohydrate esterases of family 4 which, in addition to AcXEs, contains mainly chitin deace- 3.4. Conclusions and further considerations tylases [http://afmb.cnrs-mrs.fr/CAZY/index.html].

In conclusion, the present study points to an important role of OH-groups at position 3 or 2 in deacetylation of 2,4- Acknowledgements and 3,4-di-O-Ac-Me-h-Xyl at positions 2 or 3, respectively. The use of 2-deoxy-, 3-deoxy-, 2-deoxy-2-fluoro- and 3- This work was supported by a grant from the Slovak deoxy-3-fluoro analogues of the above diacetates as AcXE Grant Agency for Science VEGA No. 2-7136/20. P. Biely 88 P. Biely et al. / Biochimica et Biophysica Acta 1622 (2003) 82–88 thanks USDA authorities for making possible his tenure a,h-D-glucopyranosides by Candida lipase-catalyzed hydrolysis, Bio- at NCAUR as a visiting scientist and for financial support org. Med. Chem. Lett. 4 (1994) 1629–1632. [10] P. Chla´dek, M. Sprinzl, The 3V-end of tRNA and its role in protein of the work. The authors thank Dr. Claude Dupont for biosynthesis, Angew. Chem., Int. Ed. Engl. 24 (1985) 371–391. the sample of acetylxylan esterase from Streptomyces [11] P. Bhuta, G. Kumar, P. Chla´dek, Elongation factor Tu.ribosome de- lividans and Drs. D. Weisleder and Juraj Alfo¨ldi for NMR pendent guanosine 5V-triphosphate hydrolysis: elucidation of the role measurements. of the aminoacyl transfer ribonucleic acid 3V-terminus and site(s) in- volved in the inducing of the guanosinetriphosphatase reaction, Bio- chemistry 21 (1982) 899–905. [12] D. Ghosh, M. Sawicki, P. Lala, M. Erman, W. Pangborn, J. Eyzaguirre, References R. Gutie´rrez, H. Jo¨rnvall, D.J. Thiel, Multiple conformations of cata- lytic serine and histidine in acetylxylan esterase at 0.90 A˚ , J. Biol. [1] P. Biely, J. Puls, H. Schneider, Acetyl xylan esterases in fungal cellu- Chem. 276 (2001) 11159–11166. lolytic systems, FEBS Lett. 186 (1985) 80–84. [13] N. Hakulinen, M. Tenkanen, J. Rouvinen, Three-dimensional struc- [2] M. Sundberg, K. Poutanen, Purification and properties of two acetyl- ture of the catalytic core of acetylxylan esterase from Trichoderma xylan esterases of Trichoderma reesei, Biotechnol. Appl. Biochem. 13 reesei: insight into the deacetylation mechanism, J. Struct. Biol. 132 (1991) 1–11. (2000) 180–190. [3] L. Christov, B.A. Prior, Esterases of xylan-degrading microorganisms: [14] M. Mastihubova´, P. Biely, A common access to 2- and 3-substituted production, properties, and significance, Enzyme Microb. Technol. 15 methyl h-D-xylopyranosides, Tetrahedron Lett. 42 (2001) 9065–9067. (1993) 460–475. [15] F. Shareck, P. Biely, R. Morosoli, D. Kluepfel, Analysis of DNA [4] G. Williamson, P.A. Kroon, C.B. Faulds, Hairy plant polysaccharides: flanking the xlnB locus of Streptomyces lividans reveals genes encod- a close shave with microbial esterases, Microbiology 144 (1998) ing acetyl xylan esterase and the RNA component of P, 2011–2023. Gene 153 (1995) 105–109. [5] P. Biely, G.L. Coˆte´, L. Kremnicky´, D. Weisleder, R.V. Greene, Sub- [16] C.N. Dupont, N. Daigneault, F. Shareck, R. Morosoli, D. Kluepfel, strate specificity of acetylxylan esterase from Schizophyllum com- Purification and characterization of an acetylxylan esterase produced mune: mode of action on acetylated carbohydrates, Biochim. by Streptomyces lividans, Biochem. J. 319 (1995) 881–886. Biophys. Acta 1298 (1966) 209–222. [17] R. Lo´pez, E. Montero, F. Sa´nchez, J. Can˜ada, A. Ferna´ndez-Mayoralas, [6] P. Biely, G.L. Coˆte´, L. Kremnicky´, R.V. Greene, C. Dupont, D. Kluep- Regioselective of alkyl h-D-xylopyranosides by use of fel, Substrate specificity and mode of action of acetylxylan esterase lipase PS in organic solvents and applications to the chemoenzymatic from Streptomyces lividans, FEBS Lett. 396 (1996) 257–260. synthesis of oligosaccharides, J. Org. Chem. 59 (1994) 7027–7032. [7] P. Biely, G.L. Coˆte´, L. Kremnicky´, R.V. Greene, Differences in cata- [18] M. Bounias, N-(1-naphtyl)ethylenediamine dihydrochloride as a new lytic properties of acetylxylan esterases and non-hemicellulolytic ace- reagent for nanomole quantification of sugars on thin-layer plates tylesterases, in: H.J. Gilbert, G.J. Davis, B. Henrissat, B. Svensson by a mathematical calibration process, Anal. Biochem. 106 (1980) (Eds.), Recent Advances in Carbohydrate Bioengineering, Royal So- 291–295. ciety of Chemistry, Cambridge, UK, 1999, pp. 73–81. [19] R.U. Lemieux, A.A. Pavia, Substitutional and solvation effects on [8] P. Biely, G.L. Coˆte´, L. Kremnicky´, R.V. Greene, M. Tenkanen, Action conformational equilibria. Effects on the interaction between oppos- of acetylxylan esterase from Trichoderma reesei on acetylated methyl ing axial oxygen atoms, Can. J. Chem. 47 (1969) 4441–4446. glycosides, FEBS Lett. 420 (1997) 121–124. [20] A. Bax, M.F. Summers, 1H and 13C Assignments from sensitivity-en- [9] K.-F. Hsiao, H.-J. Lin, D.-L. Leu, S.-H. Wu, K.-T. Wang, Kinetic hanced detection of hetero nuclear multiple-bond connectivity by 2D study of deacetylation and acetyl migration of peracetylated 1-methyl multiple quantum NMR, J. Am. Chem. Soc. 108 (1986) 2093–2096.