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-amylase enzymes
. Jane
ek3
1 Microbial Physiology Research Group, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, The Netherlands
2 Centre for Carbohydrate Bioprocessing, TNO-University of Groningen, Haren, The Netherlands
3 Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia
4 Business Unit Food and Biotechnology Innovations, TNO Quality of Life, Groningen, The Netherlands
Correspondence
M. J. E. C. van der Maarel
m.j.e.c.van.der.maarel{at}rug.nl
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ABSTRACT |
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-amylases are well-characterized extracellular enzymes that are classified into glycoside hydrolase subfamily GH13_1. This study describes the identification, and phylogenetic and biochemical analysis of novel intracellular fungal -amylases. The phylogenetic analysis shows that they cluster in the recently identified subfamily GH13_5 and display very low similarity to fungal -amylases of family GH13_1. Homologues of these intracellular enzymes are present in the genome sequences of all filamentous fungi studied, including ascomycetes and basidiomycetes. One of the enzymes belonging to this new group, Amy1p from Histoplasma capsulatum, has recently been functionally linked to the formation of cell wall -glucan. To study the biochemical characteristics of this novel cluster of -amylases, we overexpressed and purified a homologue from Aspergillus niger, AmyD, and studied its activity product profile with starch and related substrates. AmyD has a relatively low hydrolysing activity on starch (2.2 U mg–1), producing mainly maltotriose. A possible function of these enzymes in relation to cell wall -glucan synthesis is discussed.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-Amylases are widely occurring enzymes which hydrolyse the -(1,4)-glycosidic bonds in starch and glycogen, producing short maltooligosaccharides and maltose. Based on sequence similarity, most -amylases (EC 3.2.1.1) are classified in glycoside hydrolase (GH) family 13, although some -amylases originating from extremophilic organisms belong to family GH57 (Henrissat, 1991
; Henrissat & Bairoch, 1996) (see also the CAZy website, http://www.cazy.org). Based on a phylogenetic analysis of 1691 different members of the GH13 family, the family has recently been divided into 35 subfamilies, all acting on -glycosidic bonds (Stam et al., 2006). Several of these subfamilies display -amylase specificity, but many other enzyme reaction specificities are also represented. The tertiary structure of these enzymes is characterized by a (β/)8 barrel containing four highly conserved amino acid regions that form the active site (MacGregor et al., 2001), but the overall sequence similarity can be as low as 10 % and only a catalytic triad of amino acids is conserved invariantly (Machovic & Janecek, 2003). The shared (β/)8 barrel structure and catalytic mechanism within GH13 enzymes are believed to represent a common evolutionary origin (Kuriki & Imanaka, 1999; Janecek, 1997). The presence of the four conserved regions and a common secondary and tertiary structure allow construction of alignments and phylogenetic studies within the family. The phylogeny of -amylases is generally in agreement with their origin, e.g. all fungal -amylases are more related to each other than to the -amylases originating from plants or animals. -Amylases from bacteria, however, are scattered over several clusters, which group with animal, plant or fungal -amylases, or form a separate branch (Janecek, 1994).
Several -amylases from yeasts and fungi have been studied previously (see e.g. Steyn et al., 1995; Matsuura et al., 1984; Moreira et al., 2004; Boel et al., 1990). In all cases, these enzymes are secreted into the extracellular environment, where they are involved in degradation of starch and glycogen into small oligosaccharides, which can be imported into the cells to serve as energy and carbon sources. The expression levels of extracellular starch-degrading enzymes are generally increased during growth in the presence of maltose or isomaltose. This system is particularly well studied in aspergilli, in which expression of these enzymes is regulated by AmyR (Petersen et al., 1999; Tani et al., 2001; Nakamura et al., 1997).
Some recent studies have shown that different fungal GH13 enzymes may be involved in formation and/or modification of -glucans in fungal cell walls, rather than in starch degradation. The fungal cell wall is usually made up of chitin, β-glucan, -glucan, galactomannan and attached cell wall proteins (Klis et al., 2002; Beauvais & Latgé, 2001). Generally, -glucan in fungal cell walls is of the -(1,3) type with a small percentage of -(1,4) glycosidic bonds (Grün et al., 2005). Additionally, an -glucan with alternating -(1,3)/(1,4) glycosidic bonds (nigeran) has been identified in Aspergillus niger and some other ascomycete species (Barker & Carrington, 1953; Woranovicz-Barreira et al., 1999). It is generally believed that these fungal -glucans are produced by -glucan synthases, although this has never been demonstrated directly. These transmembrane enzymes contain two catalytic domains. The C-terminal, intracellular domain exhibits similarity to members of glycosyltransferase family 5 (Coutinho et al., 2003), and probably uses UDP-glucose to produce a glucan chain. The N-terminal, extracellular domain has resemblance to GH13 family enzymes and is thought to be involved in the coupling of extruded glucan chains (Grün et al., 2005; Hochstenbach et al., 1998). Two novel types of GH13 family homologues have recently been shown to play a role in fungal -glucan formation. The first of these is Aah3p, a glycosylphosphatidylinositol (GPI)-anchored protein identified in the fission yeast Schizosaccharomyces pombe (Morita et al., 2006). A knockout of the corresponding gene caused an aberrant cell shape and hypersensitivity towards cell wall-degrading enzymes, indicating a role for the Aah3p protein in cell wall integrity. The biochemical characterization of two homologous GPI-anchored proteins from A. niger (AgtA and AgtB) has revealed that they have 4--glucanotransferase activity on maltooligosaccharides and starch (van der Kaaij et al., 2007). A second type of GH13 enzyme with a role in cell wall formation is Amy1p from Histoplasma capsulatum, a close relative of the aspergilli (James et al., 2006). In this pathogenic, dimorphic fungus, -(1,3)-glucan is critical for virulence (Rappleye et al., 2004). A functional knockout strain of Amy1p, a putative intracellular -amylase, completely loses the ability to form cell wall -(1,3)-glucan and has attenuated virulence (Marion et al., 2006). No biochemical characterization of Amy1p has been reported, to our knowledge.
The recent publication of the genome sequences of four aspergilli (Machida et al., 2005; Pel et al., 2007; Nierman et al., 2005; Galagan et al., 2005) allowed the identification of all -amylase homologues in these species. In an initial analysis (Pel et al., 2007), it became apparent that these Aspergillus species encode several novel putative GH13 enzymes with relatively low similarity to the known extracellular fungal -amylases. Some of these proteins are, however, highly homologous to Amy1p from H. capsulatum. In the present study, we have identified homologues of Amy1p in aspergilli and other fungi, and performed sequence analysis as well as phylogenetic analyses on this group of novel fungal -amylases. This information is combined with the heterologous expression, purification and characterization of one of these enzymes, AmyD from A. niger, to gain a first-time insight into the biochemical properties of a representative of this group of novel fungal enzymes.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Previously identified genes with locus tags An01g13610 (AmyD) and An09g03110 (AmyE) from this database were used as query sequences in BLAST searches (Altschul et al., 1997) in all non-redundant GenBank CDS translations, RefSeq Proteins, PDB and SWISS-PROT. The possible presence of a signal peptidase cleavage site or the possibility of non-classical secretion was analysed by using the web-based search tools SignalP and SecretomeP (http://www.cbs.dtu.dk/services/) (Bendtsen et al., 2004, 2005).
Sequence alignment and evolutionary tree.
A set of sequences encoding -amylases was retrieved from GenBank (Benson et al., 2006) and SWISS-PROT (Wu et al., 2006) (Table 1). The set was supplemented with the 12 fungal sequences identified as described above, and three additional sequences: Amy1p from H. capsulatum (Marion et al., 2006), and AgtA (An09g03100) and AgtB (An12g02460) from A. niger. The alignment strategy was based on the approach described by Da Lage et al. (2004). In brief, (i) the best conserved regions, the β1, β2, β3, β4, β5, β7 and β8 strands of the catalytic (β/)8 barrel and region V of domain B (Janecek, 2002), were identified in each sequence; (ii) the segments preceding and succeeding the regions around strands β1 and β8, respectively, were cut off; (iii) the shortened sequences (amino acids 34–435 in AmyD) were aligned with the CLUSTAL W program (Thompson et al., 1994); (iv) the identified conserved sequence regions were aligned manually, if necessary; and (v) the remaining parts of the alignment (between the regions) were manually tuned where applicable. The evolutionary tree was calculated with the neighbour-joining method (Saitou & Nei, 1987) implemented in the CLUSTAL X package (Jeanmougin et al., 1998) using the final alignment including the gaps; the number of bootstrap trials used was 1000. The tree was displayed with the TreeView program (Page, 1996).
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). Escherichia coli TOP10 (Invitrogen) was used for transformation and amplification of recombinant DNA. E. coli BL21 STAR (DE3) (Invitrogen) was used for protein production. A synthetic gene with the coding sequence of An01g13610 was made by Geneart and cloned into pDONR221 using two ClaI restriction sites. The construct pDONR221-An01g13610 was subsequently used to make the final construct pDEST17-An01g13610 according to the manufacturer's instructions. All intermediate steps in the production of the construct were checked by restriction analysis, and the final construct was checked by sequencing (GATC Biotech). As a positive control for cloning and purification, and a negative control for enzyme activity assays, we also cloned the gene encoding β-glucuronidase (gus) into pDEST-17 according to the manufacturer's instructions.
Protein production and purification.
E. coli BL21 STAR (DE3) transformed with pDEST17-An01g13610 or pDEST17-gus was grown in LB medium (Ausubel et al., 1987) containing 100 µg ampicillin ml–1 at 16 °C until an OD600 of 0.4 was reached. Expression was induced by the addition of 1 mM IPTG and cultures were grown until OD600 was 0.8–1.0. Cells were harvested by centrifugation (10 min, 5000 g, 4 °C) and washed with 50 mM Tris-HCl buffer (pH 8). Cell pellets were resuspended in binding buffer (50 mM Tris-HCl buffer, pH 8, containing 500 mM NaCl, 10 mM imidazole and 5 mM β-mercaptoethanol). Cell-free extracts were produced by sonication of the resuspended cells (8x15 s with 40 s intervals, on ice) and subsequent centrifugation (20 min, 4 °C, 10 000 g). The cell lysate was applied to washed nickel-nitriloacetate (Ni-NTA) column material (Qiagen) and incubated for 2 h at 4 °C. After washing the column material with binding buffer, His-tagged proteins were eluted with 50 mM Tris-HCl buffer, pH 8, containing 250 mM NaCl, 1 mM β-mercaptoethanol and 100 mM imidazole. At each stage of protein purification, the amount of protein was measured using the Bradford method with reagents from Bio-Rad, and purity was checked by SDS-PAGE analysis (Laemmli, 1970). The enzyme was concentrated over a YM10 filter (Millipore) and stored at –20 °C in Na-barbital buffer (pH 6.5) containing 15 % glycerol, v/v. Na-barbital buffer contained 28.5 mM sodium acetate, 28.5 mM Na-barbital and 116 mM NaCl, and was brought to the desired pH by addition of HCl. After Ni-NTA purification, AmyD was used in biochemical assays for a maximum of 4 days.
Analysis of enzyme activity.
Substrates were obtained from Sigma-Aldrich, except for the following: nigerotriose was purchased from Dextra laboratories, nigerose was a kind gift from Nihon Shokuhin Kako, an -(1,3)-glucan isolated from Aspergillus nidulans was a kind gift from Dr B. J. Zonneveld (Leiden University) and a Lactobacillus reuteri exopolysaccharide (Kralj et al., 2004a) was supplied by Dr S. Kralj (University of Groningen). Starch, amylopectin and amylose type III (all from Sigma-Aldrich), used in activity assays, all originated from potato; glycogen originated from oysters.
The standard reaction conditions used to measure hydrolysing activity were as follows: the enzyme was incubated with 0.2 % (w/v) potato starch (or another substrate) in Na-barbital buffer (pH 6.5) at 37 °C. Reactions were performed in a total volume of 350 µl. Samples of 50 µl were taken from the reaction every 3 min, diluted in 50 µl Na-barbital buffer (pH 6.5) and subsequently used for determination of reducing ends using the bicinchoninic acid method (Meeuwsen et al., 2000). Six samples were taken from each reaction and all reactions were performed at least in duplicate. The amount of enzyme added depended on the batch, but generally 1 µg was added, representing between 2x10–3 and 2.5x10–3 U, with 1 U defined as the amount of enzyme producing 1 µmol reducing ends min–1. In all assays, reactions with 5 µg Ni-NTA-purified Gus were included to check for background activity. Relative enzyme activities under different conditions were assayed with the same batch of enzyme. The pH optimum was determined by performing the standard reaction at pH values between 4 and 8.5 in Na-barbital buffer. Temperature stability of AmyD was determined using the standard test for starch hydrolysis, performed with enzyme diluted in 10 µl Na-barbital, pH 6.5, incubated for 10 min at different temperatures. The Km value of AmyD for starch in the hydrolysis reaction was determined by measuring its activity with eight different concentrations of starch, varying between 0.01 and 1 % (w/v) under standard conditions in triplicate. The effect of NaCl was measured by addition of various concentrations of NaCl (between 50 mM and 0.5 M) to the standard reaction. The hydrolysis of substrates other than potato starch was determined as described above. Appropriate calibration curves were included for every measurement.
Enzymic reactions for qualitative analysis were performed as follows. Standard reactions were performed in a total volume of 10 µl, containing 20 mM disaccharide or oligosaccharide substrate, or 0.2 % polysaccharide substrate, or a combination of these, in Na-barbital (pH 6.5). Additional reactions with 100 or 500 mM maltoheptaose were performed in the same way. AmyD enzyme (1 µg) representing approximately 2x10–3 U, or 5 µg Ni-NTA-purified Gus, was added to the reaction mixture and incubated for 30 min at 37 °C. A total of 2.4 µl of reaction product was spotted on a TLC plate (Silica gel 60 F254, Merck) and after drying the plate was run for 6 h in a small amount of running buffer (butanol/ethanol/Milli-Q water, 5 : 5 : 3, v/v). After running, the plate was dried and sprayed with 50 % sulphuric acid in methanol and developed for 10 min at 110 °C.
Samples for HPLC (Dionex) analyses were prepared as follows. Reactions were performed in 1 ml 1 % amylopectin (w/v) or 1 % amylose (w/v) in Na-barbital buffer (pH 6.5) at 37 °C. AmyD enzyme (2 µg), representing approximately 4x10–3 U, or 10 µg Ni-NTA-purified Gus, was added to the reaction and samples (250 µl) were taken after 0, 10 and 60 min of incubation. Samples were diluted in 1250 µl 90 % DMSO and subsequently used for HPLC analysis performed as described previously (Kralj et al., 2004b).
Activity staining of AmyD was performed by running 2 µg Ni-NTA-purified protein on SDS-PAGE gels containing 10 % polyacrylamide and 0.12 % (w/v) amylopectin. The protein samples were not boiled and neither were denaturing components added to the loading buffer, so as to preserve enzymic activity. After separation, the gel was washed and incubated in Na-barbital buffer (pH 6.5) at 37 °C for 18 h, and subsequently stained with diluted iodine solution. Iodine stains the amylopectin in the gel purple, except in places where it is degraded by -amylase activity. Subsequently, the gel was washed and used for the staining of 6xHis-tagged proteins with the InVision staining method (Invitrogen) according to the manufacturer's instructions. Afterwards, the same gel was stained with Biosafe Coomassie and silver stain plus (both from Bio-Rad).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-amylases, distantly related to extracellular fungal -amylases (Pel et al., 2007). A BLASTP search with these two sequences was performed in the available databases (during November 2006), yielding nine fungal homologues from Aspergillus oryzae, Aspergillus fumigatus, A. nidulans, Neurospora crassa and Magnaporthe grisea. A more distantly related homologous sequence from the genome of Cryptococcus neoformans (Loftus et al., 2005) was also included. This set of predicted proteins, combined with the A. niger AmyD and AmyE proteins and Amy1p from H. capsulatum (Marion et al., 2006), was used for a detailed sequence comparison.
In A. niger, A. oryzae, A. nidulans and M. grisea, the genes encoding the Amy1p homologues are part of a small cluster of genes which are predicted to be involved in production of cell wall -glucan. Apart from an Amy1p homologue, these clusters contain genes encoding an -glucan synthase and a novel type of GPI-anchored GH13 family enzyme. A homologue of these GPI-anchored enzymes in S. pombe has recently been identified as important for cell wall -glucan synthesis (Morita et al., 2006). The retrieved protein sequences of seven out of the 12 Amy1p homologues were missing one or more of the highly conserved regions specific to the GH13 family (Janecek, 2002; Kuriki & Imanaka, 1999). After careful analysis of the original gene sequences, it appeared that the prediction of introns in these genes was not correct. The intron prediction was corrected in compliance with intron consensus elements (Kupfer et al., 2004), thereby restoring full-length protein sequences containing all conserved GH13 family residues and regions. In gene EEA30628.1 from N. crassa, the positions of two expected introns could not be predicted with certainty and thus no correction was made. Although the protein sequence was extended, it could nevertheless be used in alignments because no frameshift occurred within the sequence extensions. In A. oryzae BAE58539.1 the third conserved region could not be restored due to a frame shift, probably caused by a sequencing error. The sequence was nevertheless restored by detailed comparison with the homologous gene from another A. oryzae sequencing project (Uniprot accession number AB078784). None of the (corrected) protein sequences was predicted to have an N-terminal signal for secretion. Two proteins were slightly above the threshold level in the analysis for non-classical secretion (EAA33974.1 and An09g03110).
The 13 (corrected) protein sequences together with two novel -glucanotransferases from A. niger (An-AgtA and An-AgtB) were aligned with 25 -amylases from representative taxa of the three kingdoms of life: bacteria, archaea and eukarya. The alignment that spanned the entire catalytic (β/)8 barrel, including domain B (the β3
3 insertion), was used for calculating the phylogenetic tree (Fig. 1). The tree clearly shows the high mutual similarity of the 13 novel GH13 proteins described in this study, and their high similarity to a group of bacterial -amylases of the liquefying type recently grouped in subfamily GH13_5, represented here by proteins from Bacillus stearothermophilus (Bacst in Fig. 1) and Streptococcus mutans (Stcmu) (Stam et al., 2006). For example, the similarity of AmyD to the bacterial proteins Bacst and Stcmu was 51–56 %, while its similarity to the archaeal proteins included was 33 %. Interestingly, a protein sequence from C. neoformans (Crcne), a basidiomycete, clustered between the GH13_5 proteins from ascomycete fungi and bacteria (Fig. 1). The 13 intracellular fungal proteins are clearly unrelated to all previously identified extracellular fungal -amylases grouped presently in subfamily GH13_1, represented by A. niger acid amylase (Aspni) and A. oryzae TAKA-amylase (Aspor). The recently identified -glucanotransferases AgtA and AgtB cluster with the extracellular fungal -amylases, rather than with the intracellular group. The bacterial -amylases form several clusters in the tree, reflecting their sequence similarities to enzymes from fungi, plants or animals, as described previously (Janecek, 1994; Janecek et al., 1999; Da Lage et al., 2004).
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-amylases share several sequence features with the bacterial enzymes in the GH13_5 family. These features are, or may be, invariant among the intracellular fungal enzymes and the related bacterial enzymes, but in most cases have no (conserved) equivalent in the other -amylases studied here (Fig. 2). These specific sequence features include: (i) histidine (His57, AmyD numbering) in the region flanking the strand β2 from the N terminus; (ii) arginine (Arg60) and cysteine (Cys82) flanking strand β2 at the C terminus, which, although occurring highly specifically in most fungal GH13_5 enzymes, are absent from the C. neoformans sequence representing the basidiomycete fungi; (iii) an almost invariant leucine residue preceding the conserved NH in conserved region I, at the end of the β3-strand region (136_DAVLNH); (iv) two aromatic residues succeeding the catalytic glutamate proton donor in conserved region III (around strand β5) (297_EYWR); and (v) a cysteine and a leucine in the region covering the β8 strand (392_GQPCIFWGDL), which are invariant in all fungal proteins but only partly conserved among the related bacterial sequences. In addition to the catalytic (β/)8 barrel, domain B is also highly specific to the intracellular fungal enzymes and the bacterial enzymes in the GH13_5 family. With approximately 105 amino acids, this domain is longer than typically found in fungal -amylases in the GH13_1 family, with a B domain of around 65 amino acids.
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-amylases. PCR with specific primers did not result in retrieval of amyD or amyC (An09g03110) cDNA from cDNA libraries constructed from A. niger strain N402 grown on starch or inulin (van der Kaaij et al., 2007). Therefore, a synthetic gene encoding the predicted coding sequence of An01g13610 was made, and codon usage was optimized for expression in E. coli. Production of AmyD in E. coli was only observed when the host was grown at 16–18 °C, and the yield was relatively low (maximum yield 0.3 µg ml–1). The Ni-NTA-purified protein was incubated with starch, amylopectin and various di- and oligosaccharides to determine its enzymic activity. The main initial product formed from starch and amylopectin was maltotriose, followed by maltose and several larger maltooligosaccharides, mainly with a degree of polymerization (DP) of 6–9, as determined by HPLC analysis (Fig. 3). Maltooligosaccharides with a minimum length of five anhydroglucose units were hydrolysed, while no activity was observed on maltose, maltotriose and maltotetraose (Fig. 4). The specific activity on starch was 2.2±0.3 µmol reducing ends mg–1 min–1. Starch-hydrolysing activity in the negative control, resulting from endogenous E. coli -amylase activity, was not detectable.
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-amylase activity and the 6xHis tag (Fig. 5). No -amylase activity was detected in the control, while two activity spots were detected in the AmyD sample. These two activity bands were repeatedly co-purified upon Ni-NTA purification and subsequent anion-exchange purification (results not shown). The upper spot coincided with the main protein band obtained after Ni-NTA purification, which also stained for the 6xHis-tag. The lower band apparently represented a differently folded, but more active form of the protein. The protein concentration in this spot was too low for detection with the InVision 6xHis-tag staining method. It should be noted that the protein samples used for this SDS-PAGE were not denatured and therefore the size of the proteins cannot be derived directly from their position in relation to the marker proteins.
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-(1,6) glycosidic bonds], A. nidulans -(1,3)-glucan, nigeran [a glucan with alternating -(1,3) and -(1,4) glycosidic bonds] and L. reuteri exopolysaccharide [containing -(1,3) and -(1,6) glycosidic bonds (Kralj et al., 2004a)]. None of these substrates was hydrolysed by AmyD.
The hydrolysis reaction of AmyD on starch and similar substrates was studied in more detail. This showed that AmyD was active towards starch and amylose, but had less activity towards amylopectin, and almost no activity towards glycogen (Table 2). The Km for potato starch in the hydrolysis reaction was between 0.02 and 0.05 % starch (w/v). Addition of 0.1, 1 or 10 mM CaCl2 or 1 mM EDTA did not have a significant effect on the rate of starch hydrolysis (Table 3). Analysis of hydrolysis at different pH values showed that the enzyme had a very broad pH optimum, with highly comparable activity in the range between pH 5 and 7.5, and a slight optimum between pH 5.5 and 6.5 (Fig. 6). Within this whole pH range, maltotriose was the dominant reaction product formed (results not shown). In the absence of substrate, stability of the enzyme at increased temperatures was rather poor: activity was lost rapidly at temperatures above 35 °C (Fig. 7). Addition of 1 mM CaCl2 did not have a significant effect on heat resistance. In the presence of substrate, however, the enzyme was much more stable, as there was no significant decrease of activity during the incubations with starch, measured at 37 °C over 15 min. Addition of various amounts of NaCl had a negative effect on enzyme activity, leading to approximately 75 % activity in the presence of 500 mM NaCl, compared to the standard reaction conditions (Table 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), a novel group of -amylases has been observed with very low similarity to all other fungal -amylases described thus far. Proteins belonging to this new fungal -amylase group characteristically have high mutual sequence similarity and a predicted intracellular location. According to the recent division of GH13 family enzymes into subfamilies, the extracellular fungal -amylases are classified into group GH13_1. We conclude that the novel intracellular fungal -amylases described here are members of subfamily GH13_5, displaying, within the frame of the entire -amylase GH13 family, the highest sequence similarity to the bacterial enzymes previously assigned to this subfamily (Stam et al., 2006). Their close evolutionary relationship was confirmed by the existence of several amino acids specifically conserved within this subfamily (Fig. 2
). However, these did not include amino acids which are known to be predictive for certain enzyme reaction or product specificities within the GH13 family, such as hydrolysis of -(1,6) glycosidic bonds or -glucanotransferase activity (as reviewed by MacGregor et al., 2001). The specific functions of these conserved amino acid residues thus remain to be determined.
The 13 protein sequences subjected to sequence analysis in this paper can be considered to be examples of an undoubtedly much larger group of fungal -amylases. Homologous proteins have been identified in the genome sequences of the ascomycetes Botrytis cinerea, Chaetomium globosum and Sclerotinia sclerotiorum (http://www.broad.mit.edu/annotation/fungi/fgi/index.html). In many fungal species, the organization of the genes encoding the GH13_5 enzymes is highly conserved, strengthening the hypothesis that these enzymes have the same, or strongly related, physiological functions.
The phylogenetic analysis of the fungal sequences has shown that these -amylases are positioned on a common branch with a group of bacterial -amylases previously described as being related to plant -amylases (Da Lage et al., 2004). The similarity between the two groups implies an evolutionary relationship, as observed previously for a cluster of bacterial enzymes with similarity to animal -amylases, suggested to be a result of horizontal gene transfer (Da Lage et al., 2004). The bacterial -amylases belonging to subfamily GH13_5 include enzymes from Bacillus and Cytophaga species (Yuuki et al., 1985; Jeang et al., 2002; Kanai et al., 2004), some of which are thermostable (Kim et al., 2000) and used in industry (Guzman-Maldonado & Paredes-Lopez, 1995). Several of these bacterial enzymes are known to produce oligosaccharides of specific lengths from starch, including, for example, a maltohexaose-forming -amylase from Bacillus (Kanai et al., 2004). To analyse whether similar properties are also encountered in the related fungal enzymes, we produced and characterized a randomly chosen homologue from this cluster, AmyD from A. niger. The biochemical analysis showed that, like the related bacterial enzymes, AmyD mainly produces maltotriose from starch, amylose and amylopectin. Like other -amylases, AmyD displayed relatively low activity on highly branched substrates such as glycogen. Increased stability or activity in the presence of Ca2+, often encountered in -amylases (see e.g. Boel et al., 1990; Nielsen et al., 2003), was not observed for AmyD. In the 3D structure of bacterial GH13_5 family enzymes, a triad of metal ions (Ca2+ – Na+ – Ca2+) has been observed between domain A and domain B, and an additional Ca2+ ion has been located between domain A and domain C (Davies et al., 2005; Machius et al., 1998; Brzozowski et al., 2000). The amino acids interacting with these Ca2+ ions are only partly conserved among the fungal GH13_5 family enzymes; the presence of bound Ca2+ ions in these enzymes therefore appears less likely.
Ni-NTA-purified AmyD showed a maximum starch-hydrolysing activity of 2.5 U mg–1, which is very low compared to the activity of extracellular fungal and bacterial -amylases, which commonly have a specific activity of 100–1000 U mg–1 (see e.g. Khoo et al., 1994; Moreira et al., 2004; Dey et al., 2002). There are several possible explanations for this low activity of AmyD. First, the intracellular nature of the protein may mean that highly defined reaction conditions are needed for optimal activity. Several possibilities, such as the addition of salt or small amounts of yeast extract to supply potential cofactors, were tested, but these did not result in increased activity. Second, the substrates tested (starch and derived polymers) may not be the natural substrates for the intracellular AmyD enzyme. Although a variety of substrates with -glycosidic bonds were tested in different combinations, neither hydrolysis nor transglycosylation reactions were observed to occur on any of these substrates. Therefore, it appears likely that the observed hydrolysis of maltooligosaccharides is the natural reaction of AmyD. A third explanation is that a high AmyD activity is not needed for its physiological function in the cell. The data available on specific activities all relate to fungal -amylases acting in the extracellular environment. The latter enzymes have been selected for their ability to rapidly hydrolyse substrates, in order to minimize the chances for competing organisms to use the same carbon source. Therefore, a comparison of the activity levels of intra- and extracellular -amylase enzymes is not appropriate.
A possible physiological function for the fungal intracellular -amylases was provided by the study of the AmyD homologue Amy1p of H. capsulatum. Although the enzymic activity of this protein has not been studied, a clear link was made with the production of cell wall -glucan, as a functional knockout was completely incapable of producing -(1,3)-glucan (Marion et al., 2006). This hypothesis regarding the function of fungal GH13_5 -amylases is strengthened by the genomic organization of the encoding genes, many of which are arranged in a small cluster of genes also encoding an -glucan synthase and a GPI-anchored -glucanotransferase. Such an arrangement of genes has been observed in A. niger amyE, for example, but not for amyD, and for A. oryzae AO090003001497 but not for AO090005001193. Additionally, regulation of these -amylases by AmyR was not observed in A. niger nor in A. nidulans, contrary to what would have been expected for enzymes involved in starch degradation (X.-L. Yuan and others, unpublished results; Nakamura et al., 2006).
Recently, the first detailed structural analysis of fungal cell wall -glucan has been performed using S. pombe (Grün et al., 2005). On the basis of these data, the authors proposed a model which suggests that a small molecule consisting of -(1,4)-linked glucose residues acts as a primer for the formation of longer, -(1,3)-linked polymers by -glucan synthases. As proposed by Marion et al. (2006), the function of Amy1p, AmyD and related proteins could be to produce such a primer molecule. In this study, we have shown that AmyD indeed produces small maltooligosaccharides, with a preference for maltotriose, in vitro. The combination of previously published data on Amy1p, the genomic organization of fungal GH13_5 family genes, and the biochemical characterization of A. niger AmyD strongly suggest the involvement of these enzymes in cell wall -glucan formation. Further studies on the formation of cell wall -glucans in fungi, a process which is still relatively obscure, are needed to confirm this involvement and to determine the role of AmyD and other enzymes involved.
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ACKNOWLEDGEMENTS |
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Edited by: N. L. Glass
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Received 30 March 2007;
revised 21 June 2007;
accepted 10 July 2007.
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