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Keywords:

  • Cellulomonas fimi;
  • Xylanase structure;
  • NodB domain;
  • Deacetylase

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

The NodB-like domain from Cellulomonas fimi xylanase D is a member of a small family of structurally homologous proteins which includes a chitin deacetylase from Mucor rouxii, an acetylxylan esterase from Streptomyces lividans and the NodB protein from rhizobia . Functional analysis of the xylanase D NodB domain suggests that, like other members of the family, it is a deacetylase, whose function is to remove acetyl groups from acetylated xylan.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Microbial endo-β-1,4-xylanases are typically composed of multiple discrete domains joined together by linker sequences [1, 2]. In addition to one or more catalytic domains, they can contain auxiliary domains of mainly three types; non-catalytic polysaccharide-binding domains, the most common of which are cellulose-binding domains [2–6], thermostabilizing domains [7–10] and domains homologous with the NodB protein from nitrogen-fixing bacteria [2, 5, 6, 11]. A fourth type of non-catalytic domain, prevalent in xylanases that are part of an aggregated cellulase/hemicellulase complex, is the docking domain which mediates formation of the complex by binding to a receptor borne on a scaffolding protein [8]. Recent work has shown that cellulose-binding domains enhance the activity of hemicellulases against complex substrates, probably by increasing the effective enzyme concentration at the substrate surface [12]. Thermostabilizing domains have been described in xylanases from mesophilic [10] and thermophilic [7, 8] bacteria and may affect stability in a broad sense, increasing resistance against thermal inactivation and stabilising the enzymes against proteolytic attack.

The presence of NodB homologues in xylanases from three different bacteria is strongly indicative of an important role, but until now no specific function has been definitively attributed to these domains. In nitrogen-fixing bacteria, Nod proteins mediate synthesis of host-specific signal molecules which induce changes in root hair morphology prior to nodulation. Recent work has shown that NodB deacetylates the non-reducing N-acetylglucosamine residue of a range of chito-oligosaccharides [13]. This paper describes experiments carried out to define the role of a NodB-like domain in xylanase D (XylD) from Cellulomonas fimi[5, 14]. Full-length XylD containing the NodB domain released acetyl groups from acetylated xylan whereas XylDtr, containing the catalytic domain alone, did not, suggesting that the NodB domain in multidomain endoxylanases functions as a deacetylase.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

2.1Sequence analysis

The sequence of xynD[5] has been assigned the accession number X76729 in EMBL/GenBank/DDBJ nucleotide sequence data libraries.

2.2Purification of enzymes

Full-length XylD and truncated XylD (XylDtr) containing the catalytic domain alone (residues 1 to 226 of the full-length sequence) were purified from cell-free extracts of E. coli JM83 harbouring pCF9 and pJS5, respectively [5].

2.3Assays and analytical techniques

Xylanase activity was measured at 37°C in 50 mM K2HPO4, 12 mM citric acid buffer, pH 6.5. Esterase activity was assayed as described previously [15]. An enzymatic assay was used to quantify liberated acetic acid (Boehringer Mannheim; Cat. No. 148 261). One unit of enzyme activity released 1 μmol of product per min. Protein was measured by dye binding. The degree of acetylation of birchwood xylan [16] was measured following alkaline hydrolysis.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

3.1Domain structure of xylanase D

Xylanase D (XylD) from C. fimi is composed of four distinct domains separated by linkers rich in threonine and proline or glycine [5]. The N-terminal family 11 catalytic domain is followed by an internal polysaccharide-binding domain, specific for xylan. A second binding domain, specific for cellulose, is located within 90 residues at the C-terminus. Between the two binding domains is a fourth domain of unknown function that exhibits more than 30% sequence identity with rhizobial NodB proteins. A similar NodB-like domain is also present in xylanase C (XylC) from C. fimi[10], xylanase A (XylA) from Cellvibrio mixtus and xylanase E (XylE) from Pseudomonas fluorescens ssp. cellulosa[6].

3.2Homology of xylanase NodB domains with other deacetylases

Sequence comparisons indicated that the NodB-like domain of XylD displayed 32 and 54% identity with a chitin deacetylase (Cda) from Mucor rouxii[17] and an acetylxylan esterase (AxeA) from Streptomyces lividans[11], respectively. The structural similarity between deacetylases and the NodB-like domain of XylD has led us to propose that the role of this domain in XylD, and by inference other xylanases, may be to remove acetyl groups from acetylxylan.

3.3Biochemical properties of the XylD NodB domain

To address the hypothesis that the NodB domain in XylD functions as a deacetylase, full-length XylD (65 kDa) containing the NodB domain and XylDtr (23 kDa), containing the catalytic domain alone were purified and their activities against a range of substrates were assayed. Xylanase activity, measured using soluble xylan as substrate, was chosen as the basis for comparing equivalent amounts of catalytic protein. There was no apparent difference in the capacity of the two enzymes to release reducing sugar from acetylated birchwood xylan [16]; xylanase specific activities (units/mg catalytic protein) of XylD and XylDtr were 126 and 142, respectively, but were much reduced when compared with the values of 571 and 545 recorded against unacetylated soluble xylan. Full-length XylD, but not XylDtr, released acetic acid from chemically acetylated xylan in a time-dependent manner. At a fixed enzyme concentration, the release of acetic acid from chemically acetylated birchwood xylan during incubation at 37°C and pH 6.0 was dependent on substrate concentration; the Km and Vmax values measured for full-length XylD were 31.3 mg/ml and 0.5 μmol/min, respectively. At a fixed substrate concentration (40 mg/ml), the release of acetic acid from acetylated xylan by full-length XylD increased approximately linearly with increasing enzyme concentration. Neither of the enzymes exhibited activity against 4-nitrophenylacetate, 1-naphthyl esters of acetic, butyric, octanoic and dodecanoic acids, destarched wheat bran or the methyl esters of 3-methoxy-4-hydroxycinnamate, 3,5-dimethoxy-4-hydroxycinnamate and 3,4-dihydroxycinnamate. Previous work has shown that full-length XylD did not catalyse the deacylation of chitobiose [5]. These data suggest that the NodB domain present in full-length XylD, but absent from XylDtr, functions as a xylan-specific acetyl esterase.

3.4Rationale for the evolution of bifunctional xylanase/acetyl esterase

Acetyl substitution of the xylan backbone results in a substrate that is much less susceptible to depolymerisation by xylanases than xylan which has not been acetylated. Removal of acetyl groups by treatment with acetylxylan esterase can relieve the inhibition of xylanase activity [18], particularly for hardwood xylans, in which 70% of xylose residues may be acetylated. Many xylan-degrading bacteria and fungi meet the requirement for accessory acetyl esterase activity by producing, within their repertoire of hemicellulase enzymes, a specific acetylxylan esterase (see, for example, [11, 18]). Our demonstration that the NodB domain of XylD from C. fimi, and by inference the NodB domains described in other xylanases, is able to release acetic acid from chemically acetylated xylan indicates that an alternative strategy for overcoming the inhibitory effects of extensive acetylation on xylanase activity has been to evolve bifunctional enzymes in which xylanase catalytic domains are appended to NodB-like domains; the latter domains function as acetyl esterases and are specific in their action against substituted xylan. Modular xylanases of this type include XylC [10] and XylD from C. fimi, XylA from C. mixtus and XylE from P. fluorescens ssp. cellulosa[6]. The prevalence of this type of xylanase architecture suggests that the combined presence of xylanase and acetyl esterase domains in a single enzyme is beneficial for hydrolysis of the naturally occurring xylans contained in plant cell walls. In our experiments, full-length XylD containing a NodB domain and XylDtr from which the NodB domain had been removed were equally effective in releasing reducing sugar from acetylated xylan, even though only full-length XylD removed acetyl groups from the substrate. A possible reason for this lack of cooperativity between the xylanase and esterase activities of XylD is the low degree of acetylation of the xylan used. The birchwood xylan used in this investigation was acetylated with acetic anhydride and contained 10% by weight of acetic acid, which equates to a degree of substitution of 1 acetyl group for approximately every four xylose residues. This degree of acetylation rendered the substrate insoluble and diminished the xylanase activities of XylD and XylDtr, when compared with their activities against unacetylated soluble xylan, but may not represent a good model for naturally occurring acetylated xylan.

In summary, the results presented here show that the NodB domain of XylD from C. fimi functions as a deacetylase. Similarity between the NodB domains of various xylanases and the catalytic domains of Cda from M. rouxii and AxeA from S. lividans suggests that the genes encoding these functionally related enzymes have diverged from a single deacetylase gene.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

The support of the Biotechnology and Biological Sciences Research Council is gratefully acknowledged (Grant LE13/138).

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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