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Mode of Action of Acetylxylan Esterase from Streptomyces Lividans: a Study with Deoxy and Deoxy-Fluoro Analogues of Acetylated Methyl H-D-Xylopyranoside

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Mode of Action of Acetylxylan Esterase from Streptomyces Lividans: a Study with Deoxy and Deoxy-Fluoro Analogues of Acetylated Methyl H-D-Xylopyranoside Biochimica et Biophysica Acta 1622 (2003) 82–88 www.bba-direct.com Mode of action of acetylxylan esterase 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 enzyme–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 esterases (AcXEs) are components of micro- was consistent with the function of the enzymes 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 acetylation 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 Lipase 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.
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