1 Centre for Carbohydrate Bioengineering (CCB), TNO-University of Groningen, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands
2 Innovative Ingredients and Products Department, TNO-Nutrition and Food Research, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands
3 Innovative Ingredients and Products Department, TNO-Nutrition and Food Research, Rouaanstraat 27, 9723 CC, Groningen, The Netherlands
4 Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands
Correspondence
L. Dijkhuizen
L.Dijkhuizen{at}biol.rug.nl
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ABSTRACT |
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INTRODUCTION |
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GTF proteins of lactic acid bacteria share a common structure and are composed of four distinct domains. Their N-terminal end starts with: (i) a signal peptide of 3234 amino acids, followed by (ii) a highly variable stretch of 123129 amino acids, (iii) a highly conserved catalytic or sucrose-binding domain of about 1000 amino acids, and (iv) a C-terminal domain of about 500 amino acids, composed of a series of tandem repeats (Monchois et al., 1999c). We were interested in the precise roles of these various domains and repeats in the overall functioning of GTF enzymes.
Amino-acid sequence comparisons revealed that the catalytic core of GTF proteins is constituted of eight -sheets alternated with eight
-helices [a (
/
)8 barrel structure], similar to glycoside hydrolases of family 13 (
-amylase family). This family includes, for instance,
-amylase and cyclodextrin glycosyltransferase (CGTase) (van der Veen et al., 2000
). In GTFs, however, this (
/
)8 barrel structure is circularly permuted (MacGregor et al., 1996
; Devulapalle et al., 1997
). Therefore GTFs are classified in family 70 of glycoside hydrolases (http://afmb.cnrs-mrs.fr/CAZY/).
Whereas the catalytic mechanism for the -amylase family is known (McCarter & Withers, 1994
; Uitdehaag et al., 1999
), the exact catalytic mechanism of GTF enzymes remains to be elucidated (Monchois et al., 1999c
). The amino-acid residues crucial for catalysis in glucansucrases of family 70 have been identified as Asp453 (putative catalytic nucleophile), Glu491 (putative acid/base catalyst) and Asp564 (putative transition-state stabilizer) in GTFI from Streptococcus downei Mfe28 (Fig. 1
) (MacGregor et al., 1996
; Devulapalle et al., 1997
). The equivalent invariable residues in enzymes of
-amylase family 13 are Asp229, Glu257 and Asp328 (CGTase Bacillus circulans 251 numbering). In enzymes of both families, the first-mentioned Asp residue is involved in the formation of the covalent glucosylenzyme complexes (Mooser et al., 1991
; MacGregor et al., 1996
; Uitdehaag et al., 1999
). The importance of this Asp residue has also been shown for other GTFs by site-directed mutagenesis experiments (Kato et al., 1992
; Monchois et al., 1997
; Devulapalle et al., 1997
) (Fig. 1
). Based on alignments with glucansucrases from lactic acid bacteria (Monchois et al., 1999c
), putative catalytic residues in Lb. reuteri strain 121 GTFA were identified and mutated (see Results and Fig. 1
).
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The C-terminal domains of Streptococcus and Leuconostoc GTF enzymes consist of a series of different tandem repeats, which have been divided into four classes: A, B, C and D. In addition to A and C repeats, DSRS from Ln. mesenteroides NRRL-512F contains N repeats (Monchois et al., 1998b), which have not been identified in other GTFs. Alternansucrase from Ln. mesenteroides NRRL-B 1355 contains a single A repeat and distinct short repeats DG(X)4APY (Janecek et al., 2000
). Within the AD repeats, a repeating unit designated YG can be distinguished (Giffard & Jacques, 1994
). The C-terminal repeats play an important role in glucan binding (Abo et al., 1991
; Shah & Russell, 2002
; Lis et al., 1995
; Monchois et al., 1998b
).
The structure of Lb. reuteri 121 GTFA is unusual because the above-mentioned repeats are not present in its N- and C-termini. Instead, GTFA possesses a relatively large N-terminal variable domain (702 amino acids) with 5 RDV repeats. Its relatively short C-terminal domain (267 amino acids) contains 11 YG repeats only (Fig. 2A) (Kralj et al., 2002
). This raised questions about the precise role of the N- and C-terminal domains in GTFA.
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METHODS |
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Molecular techniques.
General procedures for cloning, E. coli transformation, DNA manipulation, and agarose gel electrophoresis were as described (Sambrook et al., 1989). Restriction endonuclease digestion and ligation with T4 DNA ligase were performed as recommended by the enzyme suppliers (New England Biolabs; Roche Biochemicals). Primers were obtained from Eurogentec, Seraing, Belgium. Cycle sequencing (Murray, 1989
) was performed on double-stranded DNA, using the Thermo Sequence fluorescent primer cycle sequence kit (Amersham Pharmacia Biotech). Sequence reactions were performed on the Amersham ALF-Express sequencing machine at the BioMedical Technology Center, Groningen. DNA was amplified by PCR on a DNA Thermal Cycler PTC-200 (MJ Research), using Pwo DNA polymerase (Roche Biochemicals). Fragments were isolated from agarose gels using a Qiagen gel extraction kit, following the instructions of the supplier.
Plasmid construction.
Plasmid pBGTF1, pBluescript carrying the Lb. reuteri 121 gene encoding GTFA (Kralj et al., 2002), without signal sequence and fused at the C-terminus with six histidine residues (1750 amino acids), was used. To facilitate further mutagenesis and nucleotide sequencing, the first of two EcoRV restriction sites (492 bp, 4105 bp) was altered in pBGTF1, using the megaprimer method (Sarkar & Sommer, 1990
) and the following primers: GTFpr1, 5'-GATGCATGAGCTCCCATGGACCAACAAGTTCAGCAAGCTTCC-3', containing SacI (italics) and NcoI (bold) restriction sites, and mEcoRV, 5'-GGTTGTGATGTCACGCACAATGATTTG-3', containing a mutated EcoRV site (underlined, silent mutation by change of base shown in bold). In a subsequent PCR reaction, the amplified product (
500 bp) was used as forward primer, together with GTFpr12 (5'-GTGCATTAAAGTACGTAACCAATCAGTATTTCCGG-3'), 1600 bp downstream of an XbaI restriction site (Fig. 2A
). The resulting product of 2600 bp was digested with SacI and XbaI and ligated in the corresponding sites of pBGTF1, yielding pBGTF2.
Plasmid pBGTF2 now consisted (from 5' to 3') of four cassettes of 10001500 bp: i) SacI&(NcoI)/XbaI (1001 bp), ii) XbaI/PstI (1546 bp), iii) PstI/EcoRV (1454 bp), and iv) EcoRV/(BamHI)&ApaI (1269 bp) (Fig. 2A). This plasmid was digested with PstI (2655 bp) and EcoRV (4105 bp), and the resulting fragment (1454 bp) was ligated in the corresponding sites of pBluescript II SK+ (Stratagene), yielding pBPE1500. This small construct was used for site-directed mutagenesis, sequencing, and rapid exchange (using PstI and EcoRV restriction sites) with pBGTF2 (see below). pBGTF2 was also digested with NcoI/BamHI (5257 bp) and ligated in the corresponding sites of pET15b (Novagen), yielding p15GTF2 (encoding GTFA-CHis; Fig. 2B
). This construct was used in subsequent cloning steps.
In order to create a construct without a C-terminal His tag, but with a stop codon, the following primers were used to exchange the C-terminal part of p15GTF2: SpeFor, 5'-GGGCTTTCAGATGCAACTAATCGTTGGGG-3', lying 400 bp upstream of a SpeI restriction site, and GtfARevstop, 5'-TCGATGGGCCCCGGATCCTATTATAGTTTATTTTGATCAAGCATCTTACC-3', containing BamHI (bold) and ApaI (italic) restriction sites (Fig. 2A). The resulting PCR product (
2000 bp) was digested with SpeI and BamHI (1592 bp) and ligated in the corresponding sites of p15GTF2 (see above), yielding p15GTFstop. In all cases, successful mutagenesis was confirmed by nucleotide sequencing.
Site-directed mutagenesis of putative catalytic residues of GTFA.
Plasmid pBPE1500 (see above) was used as template for mutagenesis. The QuickChange site-directed mutagenesis kit (Stratagene) was used to construct mutants D1024N, E1061Q and D1133N, using different primer pairs (Table 1A). Successful mutagenesis resulted in the appearance or disappearance of restriction sites, allowing rapid screening of potential mutants. After successful mutagenesis (confirmed by nucleotide sequencing), pBPE1500 (mutation-containing) was digested with PstI and EcoRV and ligated in the corresponding sites of pBGTF2. The resulting plasmid, pBGTF2 (mutation-containing), was digested with NcoI/BamHI, and the resulting 5·3 kb fragment was ligated into the corresponding sites of the expression vector pET15b (Novagen), yielding mutation-containing p15GTF2.
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Purification of GTFA (mutant) proteins.
Cells of E. coli BL21star (DE3) harbouring the different pET15b derivatives were harvested by centrifugation (10 min, 4 °C, 10 000 g) after 16 h of growth at 37 °C without induction. The pellet was washed with 50 mM phosphate buffer, pH 8·0. Pelleted cells were resuspended in 50 mM sodium phosphate buffer, pH 8·0, containing 250 mM NaCl, 5 mM -mercaptoethanol and 10 mM imidazole. Cells were broken by sonication (7x15 s at 7 micron with 30 s intervals), and centrifuged (10 min, 4 °C, 10 000 g). The clear lysate containing GTF activity was loaded on a Ni-NTA column (Qiagen). Binding was realized using 50 mM sodium phosphate buffer, pH 8·0, containing 250 mM NaCl, 5 mM
-mercaptoethanol and 10 mM imidazole, followed by washing with the same buffer. Elution of His-tagged protein(s) was performed using 50 mM sodium phosphate buffer, pH 8·0, with 250 mM NaCl, 1 mM
-mercaptoethanol and 200 mM imidazole. Eluted proteins were desalted with 20 mM Tris buffer, pH 8·0, using a 5 ml Hi-Trap desalting column (Amersham Pharmacia Biotech). Subsequently, the samples were purified on an ACTA prime FPLC system (Amersham Pharmacia Biotech), using a 1 ml Resource-Q column (Amersham Pharmacia Biotech) and a linear gradient of 30 ml with 1 M NaCl in 20 mM Tris buffer, pH 8·0, as eluent, at a flow rate of 1 ml min1. Proteins present in the elution peak were desalted with 25 mM sodium acetate buffer, pH 4·7, supplemented with 1 mM CaCl2, using a 5 ml Hi-Trap desalting column (Amersham Pharmacia Biotech). At each stage of the purification, the GTF-transferase activity was quantified, as previously described (van Geel-Schutten et al., 1999
). The degree of purity of the different mutants was determined by SDS-PAGE (Laemmli, 1970
).
Protein concentrations were determined by the Bradford method, using the Bio-Rad reagent and BSA as a standard (Bio-Rad). From 250 ml cultures of E. coli strains expressing full-length (wild-type or mutant) and N- or C-terminally truncated protein, approximately 0·25 and 1 mg of highly purified protein were obtained, respectively.
Glucan binding assay.
The glucan-binding ability of GTFA (deletion mutants) was measured as described by Lis et al. (1995), and as improved by Shah & Russell (2002)
. Briefly, 30 pmol of GTFA (mutant) protein in 200 µl PBS-T (20 mM sodium phosphate, pH 7·3, 150 mM NaCl, 0·05 % Tween 20) was incubated overnight at 4 °C in Ni-NTA-coated HisSorb plates (Qiagen). Proteins containing a His tag bound to the Ni-NTA; non-binding protein was removed by washing with PBS-T (four times 1 min). After incubation for 20 min with 200 µl biotin-labelled dextran (100 µg µl1; Fluka) in PBS-B (20 mM sodium phosphate, pH 7·3, 150 mM NaCl, 0·2 % BSA), which may bind to the glucan-binding domain (GBD) of the protein, washing was performed with PBS-T, as described above. Extravidinalkaline phosphatase conjugate (1/10 000; Sigma) in PBS-B, which binds to the biotin part of the labelled dextran, was added and incubated for 1 h, followed by washing with PBS-T. Subsequently, the alkaline phosphatase substrate 4-nitrophenyl phosphate (100 µl; Roche Biochemicals) was added, yielding a yellow colour upon hydrolysis by the bound alkaline phosphatase. The colour change was monitored using a SpectraMax Plus 384 plate reader (Molecular Devices).
Enzyme activity assays.
The various reuteransucrase activities were determined by measuring glucose and fructose release (enzymically) from sucrose conversion (van Geel-Schutten et al., 1999). The amount of fructose released (VF) corresponds to total enzyme activity (initial formation of leucrose or other sucrose isomers was negligible; see Results and data not shown). The amount of free glucose (VG) represents the hydrolytic activity of the enzyme. The amount of fructose minus the amount of free glucose reflects the transferase activity (VFVG). Unless indicated otherwise, reactions were performed at 50 °C in 25 mM sodium acetate buffer, pH 4·7, containing 1 mM CaCl2 and 30 nM purified (mutant) reuteransucrase enzyme. One unit of enzyme activity was defined as the release of 1 µmol of monosaccharide per min. In the case of very low activity of mutant proteins, assay conditions were modified as follows: reactions were performed with 25 times more protein, and aliquots were removed at 30 min intervals, instead of 1 min.
(i) Kinetic parameters.
Kinetic assays were performed using 24 different sucrose concentrations, ranging from 0·25 to 100 mM. Over a 6 min incubation period, samples of 25 µl were withdrawn every minute and inactivated with 2·5 µl 1 M NaOH. Curve fitting of the data was performed with the SigmaPlot program (version 8.0), using either the MichaelisMenten formula [y=(axx)/(b+x)] or the same formula with a substrate-inhibition constant [y=(axx)/(b+x+(x2/c))]. In these equations, y is the specific activity (U mg1), x is the substrate concentration (mM sucrose), a is the maximal reaction rate [Vmax (U mg1)], b is the affinity constant for the substrate [Km (mM sucrose)], and c is the substrate inhibition constant [Ki (mM sucrose)].
(ii) Effect of maltose on initial GTFA activity.
The initial rate of oligosaccharide synthesis was examined by measuring the effect of maltose on (mutant) GTFA enzyme activity, using 50 mM sucrose and 100 mM maltose. The activity was determined by measuring fructose release.
Product analysis
(i) Product spectrum from sucrose.
After complete depletion of sucrose (100 mM, 60 h at 50 °C), the concentrations of fructose, glucose and leucrose in the reaction medium of GTFA (mutants) were determined using anion-exchange chromatography (see below). The amount of fructose released (97·9 %) and leucrose (2·1 %) synthesized from sucrose corresponds to 100 %. Subtracting the free glucose (23·1 %; due to hydrolysis) from the free fructose (97·9 %) concentration allowed calculation of the yield of reuteran synthesis (74·8 %) from sucrose (data of GTFA-CHis used here as an example, see Table 4).
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To determine the degree of polymerization (DP) of unknown oligosaccharides, a BC-200 Ca2+ column (at 85 °C; 300 mmx7·8 mm; Benson Polymeric, Reno, USA) eluted with water (0·2 ml min1) was used. Detection was done by using a model 830-RI refractive index detector at 40 °C (Jasco, Tokyo, Japan).
In vitro glucan production by (mutant) GTFA enzymes and glucan structure analysis
(i) Polymer production.
Purified (mutant) enzyme preparations were incubated overnight with 146 mM sucrose, using the conditions described above under enzyme activity assays. Glucans produced were isolated by precipitation with ethanol (van Geel-Schutten et al., 1999).
(ii) Methylation analysis.
Polysaccharides were permethylated using methyl iodide and Na-Dimsyl in DMSO at room temperature (Hakomori, 1964). After hydrolysis with 2 M trifluoric acetic acid (1 h, 125 °C), the partially methylated monosaccharides were reduced with NaBD4 (Harris et al., 1984
). The mixtures of partially methylated alditol acetates obtained were analysed by GLC on a CP Sil 5 CB column (25 mx0·53 mm; Chrompack) and by GLC-MS on an RTX Sil MS column (30 mx0·25 mm; Restek) (Chaplin, 1982
; Jansson et al., 1976
).
(iii) Molecular masses of the glucans.
Molecular mass analysis was performed as described previously, using high-performance size-exclusion chromatography (HPSEC) coupled on-line with multi-angle laser light scattering (MALLS) and differential refractive index detection (Kralj et al., 2002).
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RESULTS |
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Effects of pH, temperature and metal ions on GTFA activity
In order to define the best conditions for subsequent kinetic studies, the pH and temperature optima of GTFA activity were examined. The pH optimum for the hydrolysing activity was in the range pH 4·55·5 (Fig. 3A). For the transferase activity, a pH optimum at pH 4·7 was observed. The temperature optimum for both reactions was 50 °C. At lower temperatures, activity remained relatively high (Fig. 3B
; only shown for transferase activity).
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Product analysis
(i) Product spectrum from sucrose.
After complete depletion of sucrose, GTFA-CHis showed the following product distribution: reuteran synthesis, 74·8 %; hydrolysis, 23·1 %; and leucrose (-D-glucopyranosyl-(1
5)-
-D-fructofuranoside) synthesis, 2·1 % (Table 4
). Dionex analysis showed that a minor amount of isomaltose and significant amounts of an unknown product that eluted after 35 min were synthesized (data not shown).
(ii) Oligosaccharide synthesis with maltose and isomaltose as acceptor substrates.
Dionex analysis showed that, in the presence of maltose, GTFA-CHis formed panose (-D-glucopyranosyl-(1
6)-
-D-glucopyranosyl-(1
4)-D-glucose) as the most abundant acceptor reaction product (from 100 mM sucrose and 100 mM maltose, approximately 44 mM panose was synthesized), indicating that an
-1,6 linkage was formed at the non-reducing end of maltose (Fig. 5
B). When isomaltose was used as acceptor, low amounts of isomaltotriose and isomaltotetraose (not shown in standard; elution between 3536 min) were formed, together with two abundant unknown oligosaccharide products (Fig. 5C
) with a DP of 3 (most likely isopanose,
-D-glucopyranosyl-(1
4)-[
-D-glucopyranosyl-(1
6)]-D-glucose) and 4, respectively (DP determined by HPLC, data not shown).
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Characteristics of GTFA deletion mutants
(i) Glucan binding.
GTFA-N-CHis displayed lower glucan-binding activity (GBA) than GTFA-
N-NHis (Figs 2B and 6
). GTFA-CHis showed virtually no GBA. GTFA-
N
4YG-NHis completely lacked GBA. GTFA-
N
5YG-NHis, GTFA-
N
6YG-NHis and GTFA-
N
7YG-NHis were again active, but showed gradually lower GBA (Fig. 6
). GTFA-
N
8YG-NHis and GTFA-
N
11YG-NHis were virtually inactive.
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(iii) Effect of maltose on initial mutant GTFA activity.
The stimulatory effect of maltose on GTFA-N-NHis and GTFA-
N-CHis total activity was 1·5 to 2 times lower than on wild-type. In contrast, total activity of mutants GTFA-
N-
4YG-NHis, GTFA-
N-
5YG-NHis and GTFA-
N-
6YG-NHis was stimulated 5·5, 6 and 7·5 times, respectively, by maltose (Table 3B
).
(iv) Product spectrum from sucrose.
When incubated with sucrose, mutants with C-terminal deletions showed slightly increased reuteran synthesis and (slightly) decreased hydrolysis (Table 4).
(v) Oligosaccharide synthesis with maltose and isomaltose as acceptor substrates.
In the acceptor reaction, all deletion mutants showed similar oligosaccharide yields to wild-type (Table 4). The deletion mutants produced the same oligosaccharides as wild-type (Fig. 5
and data not shown).
(vi) Molecular sizes and linkage-type analysis of glucans produced.
Compared to GTFA-CHis, the deletion mutants showed no drastic changes in the sizes and linkage types of the glucans produced (Table 5). However, the glucan products of mutants GTFA-
N
5YG-NHis and GTFA-
N
6YG-NHis showed less branching (lower percentage of both terminal and branched
-(1
4,6) glucosyl units), a higher percentage of
-(1
6) linkages, and slightly decreased molecular masses compared to wild-type enzyme and other deletion mutants (Table 5
).
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DISCUSSION |
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The GTFA pH optimum was comparable to that of other glucansucrases. Interestingly, transferase activity showed a sharp optimum around pH 4·7, whereas the hydrolysis reaction had a broader optimal pH range (Fig. 3A). This is the first demonstration of different pH profiles for these GTF-catalysed reactions. A striking feature of the recombinant GTFA-CHis protein is its high optimal temperature of 50 °C (Fig. 3B
). This high temperature optimum has also been observed for the inulosucrase and levansucrase enzymes of the same strain (van Hijum et al., 2002
, 2003
, 2004
).
Of all cations tested, Ca2+ had the most stimulating effect on enzyme activity (Table 2): hydrolysis and transferase activity were increased two and eightfold, respectively. Zn2+, Hg2+, Fe3+, Fe2+ and Cu2+ ions significantly inhibited enzyme activity. These ions (except for Hg2+, which was not tested) also inhibit the gtfB-, gtfC- and gtfD-encoded enzymes of Streptococcus mutans (Wunder & Bowen, 1999
).
Kinetics of GTFA
Kinetic analysis of GTFA-CHis revealed interesting features (Fig. 4). At high sucrose concentrations, substrate inhibition was observed for the GTFA-CHis hydrolysis reaction. GTFA-CHis clearly favours hydrolysis at low sucrose concentrations and transferase at high sucrose concentrations. The same phenomenon has otherwise been observed only for the family 13 enzyme amylosucrase from Neisseria polysaccharea (Potocki de Montalk et al., 2000
).
The GTFA affinity for sucrose in the total reaction was much higher (Km 0·9 mM) than that observed for GTFI of S. downei Mfe28 and DSRS of Ln. mesenteroides NRRL B512F (Km 49 and 26 mM, respectively; Monchois et al., 1997, 2000a
). The stimulating effect of maltose on initial GTFA velocity (three to fourfold activation of total activity) was also observed for DSRS of Ln. mesenteroides NRRL B-512F and GTFI of S. downei Mfe28 (two and fourfold stimulation of total activity, respectively; Monchois et al., 1997
, 2000a
).
Product spectrum from sucrose
Upon depletion of sucrose, GTFA formed only low levels of leucrose (2·1 % of total sucrose consumed), even when incubated together with a high (100 mM) concentration of fructose (about 5 % leucrose synthesized) (Table 4 and data not shown). GTFI of S. downei, on the other hand, produced significant amounts (24·0 %) of leucrose (Monchois et al., 2000a
, b
). GTFA synthesized more glucan (74·8 %), and was more effective in hydrolysis (23·1 %) than GTFI of S. downei Mfe28 (60·0 % and 16·2 %, respectively; Monchois et al., 2000a
). Fructose is thus a better acceptor for GTFI of S. downei Mfe28 than for GTFA of Lb. reuteri 121.
Oligosaccharide synthesis with maltose and isomaltose as acceptors
Analysis of the acceptor reaction of GTFA revealed that, with glucose and sucrose, only a small amount of isomaltose was formed (data not shown). Previous studies showed that the production of isomaltose by DSRS from Ln. mesenteroides NRRL B-512F could be improved by using high amounts of sucrose and glucose (Buchholz & Seibel, 2003).
Maltose is a good acceptor reaction substrate for DSRS of Ln. mesenteroides B-512F (Robyt & Walseth, 1978; Monchois et al., 1997
), for GTFI of S. downei Mfe28 (Monchois et al., 1999b
) and for GTFA of Lb. reuteri 121 (this study). GTFA produced panose as major product with maltose as acceptor (formation of an
-(1
6) linkage to the non-reducing end of maltose), together with low amounts of maltotriose and two unknown products (Fig. 5B
). The formation of panose has also been observed for other GTF enzymes (Monchois et al., 1996
, 1997
, 1998a
, 2000b
; Robyt, 1996
). Compared to GTFI of S. downei Mfe28 (91·3 %) and DSRS of Ln. mesenteroides NRRL-B512F (93 %; Monchois et al., 1998b
, 2000a
), the yield of oligosaccharides synthesized by GTFA-CHis with maltose as acceptor substrate (59·6 %) is lower. Maltose is thus a better acceptor reaction substrate for GTFI and DSRS than for GTFA.
When isomaltose was used as acceptor reaction substrate, two unknown products (identified by HPLC as DP 3 and DP 4, data not shown) were produced. Maltotriose or panose eluted at different time points than the DP 3 oligosaccharide; its most likely identity is isopanose. The coupling of a glucose moiety, using one of the two linkage types synthesized by GTFA, -(1
4) and
-(1
6), to the reducing end of isomaltose would have resulted in the formation of isomaltotriose (of which small amounts are indeed synthesized) or panose (not formed) (Fig. 5C
). However, various studies show that glucansucrases elongate oligosaccharides at their non-reducing end (Mukasa et al., 2000
; Dols et al., 1997
; Argüello Morales et al., 2001
; Monchois et al., 2000a
). The small amount of isomaltotriose is thus probably formed by coupling of glucose with an
-(1
6) linkage to the non-reducing end of isomaltose. Linkage specificity (inside glucans) of several glucansucrases is conserved in oligosaccharide synthesis (Dols et al., 1997
; Cote & Robyt, 1982
; Robyt & Walseth, 1978
). This suggests that the DP 3 oligosaccharide is isopanose (coupling of a glucose unit by an
-(1
4) glucosidic bond onto the non-reducing end of isomaltose). Besides the DP 3 oligosaccharide, a DP 4 oligosaccharide was also synthesized. The exact nature of both oligosaccharides will be analysed in future. However, it appears likely that reuteransucrase is capable of forming both
-(1
4) and
-(1
6) glycosidic bonds with acceptor reaction substrates (Fig. 5
).
Characteristics of catalytic mutants
Mutagenesis of the Lb. reuteri strain 121 GTFA putative catalytic residues (mutants D1024N, E1061Q and D1133N) resulted in drastically reduced total enzymic activities. D1024 of GTFA is homologous to: (1) the Asp residues identified by Mooser et al. (1991) as part of the active site of Streptococcus sobrinus GTFI and GTFS, (2) Asp451 of GTFB from S. mutans GS5, (3) Asp453 of GTFI from S. downei Mfe28 and (4) Asp551 of DSRS of Ln. mesenteroides NRRL-B512F, which were also shown to be essential for activity (Kato et al., 1992
; Monchois et al., 1997
; Devulapalle et al., 1997
; Fig. 1
). GTFA residues E1061 and D1133 correspond to residues E491 and D564 in GTFI of S. downei Mfe28. These residues are essential for activity in both GTFI and GTFA (Devulapalle et al., 1997
; this study).
Characteristics of deletion mutants
GTFA possesses a relatively large N-terminal variable domain (702 amino acids) containing 5 RDV repeats (Kralj et al., 2002). Its complete deletion had a drastic effect on enzyme kinetics. The initial transferase activity of GTFA-
N-CHis and GTFA-
N-NHis increased three to fourfold, whereas the hydrolytic activity decreased (Table 3
). Conceivably, the large N-terminal domain in the wild-type protein causes steric hindrance to the growing glucan chain, its deletion resulting in a strongly increased transferase activity. The affinities for the substrate sucrose remained similar, in both the transferase and the hydrolysis reaction (Table 3
). The data show that the large N-terminal variable domain is important for initial activity with sucrose (Table 3
), but has only small effects on the product spectrum with sucrose alone, or sucrose plus acceptor substrates (Table 4
), and on glucan characteristics (Table 5
).
The GTFA mutants with N-terminal and additional C-terminal deletions showed drastically decreased affinity for sucrose in the total (VF) and transferase (VFVG) reactions (Fig. 4B and Table 3
). In contrast, C-terminal deletions in DSRS of Ln. mesenteroides NRRL-B512F have virtually no effects on Km values with sucrose (Monchois et al., 1998b
). Sequential deletions of the YG repeats from the C-terminus onwards resulted in gradually lower activity of the mutant GTFA enzymes in all reactions (Table 3A
). The stimulatory effect of maltose increased upon deletion of C-terminal YG repeats (Table 3B
). Both phenomena have also been observed for DSRS from Ln. mesenteroides NRRL-B512F (Monchois et al., 1998b
). The increased stimulatory effect of maltose on C-terminal deletions in DSRS was explained by a change in a rate-limiting reaction step. C-terminal deletions in GTFI of S. downei Mfe28 have no effect on the distribution of glucosyl residues during mutan synthesis (Monchois et al., 1999a
). The reuterans produced by wild-type GTFA and C-terminal deletion mutants had comparable sizes and linkage-type distribution, except for GTFA-
N
5YG-NHis and GTFA-
N
6YG-NHis. The latter mutants showed less branching (lower amount of both terminal and branched
-(1
4,6) glucosyl units), a higher percentage of
-(1
6) linkages, and a slightly decreased molecular mass compared to the wild-type and other mutants (Table 5
). C-terminal deletions in GTFI of S. downei Mfe28 do not affect glucan structure (Monchois et al., 1999a
). However, deletion of six internal units (AC) of GTFG of Streptococcus gordonii also affects glucan structure (Vickerman et al., 1996
). GTFA enzyme activity could still be detected after removal of more than half (152 amino acids, 6 YG repeats) of the GBD (Table 3
). When more than six YG repeats were removed, enzyme activity was reduced drastically (Table 3
). C-terminal deletions in other glucansucrases also affect enzyme activity (Monchois et al., 1998b
; Kato & Kuramitsu, 1990
). Only in the case of GTFI of S. downei Mfe28 could the C-terminal domain be removed completely without large loss of activity (Monchois et al., 1999a
).
The C-terminal domains of glucansucrases from leuconostocs and streptococci are involved in glucan binding (Abo et al., 1991; Shah & Russell, 2002
; Lis et al., 1995
; Monchois et al., 1998b
). The A repeats found in the C-terminal domains of these GTF enzymes are not present in Lb. reuteri 121 GTFA. Instead, 11 YG repeats have been identified (Kralj et al., 2002
). Their deletion revealed that the GTFA C-terminal domain and these YG repeats are involved in glucan binding (Fig. 6
). GTFA-
N
4YG-NHis was not able to bind biotindextran, which may be due to conformational changes in this mutant protein. GTFA-CHis showed virtually no GBA. The location of the His tag (GTFA-
N-CHis also showed lower GBA than GTFA-
N-NHis), together with the presence of the large N-terminal variable domain, may block the access of the GBD to the biotindextran. We had to incubate the GTFA protein/biotindextran/extravidinalkaline phosphatase complex for several hours with the 4-nitrophenyl phosphate substrate to be able to measure GBA, whereas in a previous report with GFTI of S. downei Mfe28, incubation for a few minutes was enough to measure comparable amounts of GBA (Shah & Russell, 2002
). Reuteran, rather than biotindextran, may be the preferred ligand for the C-terminal repeats of GTFA. Nevertheless, we show for the first time that, in reuteransucrase also, an
-(1
4) synthesizing glucansucrase from a Lactobacillus strain, the C-terminal domain is involved in glucan binding. Furthermore, positions at which truncation prevented glucan binding (deletion of eight YG repeats, 100 amino acids after the end of catalytic core) were identified (Fig. 6
).
The GTFA hydrolytic activity remained upon deletion of YG repeats, whereas glucan binding and affinity for sucrose in the transferase reaction decreased. This may imply that the glucan-binding domain is involved in polymer chain growth, as previously postulated for the GTFI from S. mutans GS5 and GTFI from S. sobrinus 6715 (Kato & Kuramitsu, 1990; Abo et al., 1991
).
Conclusion
The first thorough molecular and biochemical investigation of GTFA, a GTF enzyme synthesizing linkages of the -(1
4) glucosidic type, has been carried out. This includes analysis of the kinetics of wild-type enzyme and derived N- and C-terminally truncated mutants, analysis of the acceptor reaction, and analysis of glucan binding by the C-terminal domain. Finally, three catalytically important residues have been identified and characterized.
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ACKNOWLEDGEMENTS |
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Received 18 February 2004;
revised 26 March 2004;
accepted 2 April 2004.
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