From the Institute of Protein Biochemistry, Consiglio
Nazionale delle Ricerche, Via P. Castellino 111, 80131 Naples,
Italy, the § Istituto di Chimica Biomolecolare, Consiglio
Nazionale delle Ricerche, Via Campi Flegrei 34, 80078 Pozzuoli
(NA), Italy, and the ¶ Dipartimento di Chimica Biologica
Università di Napoli "Federico II," Via Mezzocannone 16, 80134 Naples, Italy
Received for publication, November 20, 2002, and in revised form, February 4, 2003
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ABSTRACT |
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The analysis of the complete genome of the
thermoacidophilic Archaeon Sulfolobus solfataricus
revealed two open reading frames (ORF), named SSO11867 and SSO3060,
interrupted by a It has been known that carbohydrates can serve as
structural components of natural products, as energy sources, or more
interestingly, as key elements in various molecular recognition
processes. In this regard, In plants, Family 29 of glycosyl hydrolases classification
(GH29)1 groups
In the genome of this Archaeon several interrupted genes, conserved in
distantly related Archaea, have been identified, arguing that these
interruptions were not sequencing mistakes. Rather, since no multiple
interruptions could be found, a selection toward a single conserved
shift occurs, suggesting a conserved translational regulation
mechanism.2 The maintenance
of a correct reading frame is fundamental to the integrity of the
translation process; nevertheless, an increasing number of cases have
been described in which localized deviations from the standard
translational rules are used to regulate the correct expression of a
minority of genes (18). These events, named recoding, are
used to increase the diversity in gene expression or for its
regulation, and they include programmed ribosome frameshifting to a
different reading frame, ribosome hopping over nucleotides, and reading
of stop codons as sense codons (readthrough). Among recoding events,
programmed To test the hypothesis that SSO11867 and SSO3060 ORFs could express a
functional Archaeal Strain and Cultivation--
S. solfataricus
cells, strain MT4, were grown at 87 °C, pH 3.0, as reported
previously (22); instead, strain P2 was grown at 80 °C, pH 3.5, in a
minimal salts culture medium supplemented with yeast extract (0.1%),
casamino acids (0.1%), plus glucose (0.1%). Growth was monitored
spectrophotometrically at 600 nm and stopped when it reached OD
0.6-0.8.
Substrates--
All commercially available substrates were
purchased from Sigma.
The standard program used was as follows: hot start 5 min at 95 °C,
2 min at 48 °C, and 4 min at 72 °C; 10 cycles at 95 °C for
45 s, 48 °C for 1 min, and 72 °C for 4 min; 20 cycles at
95 °C for 45 s, 58 °C 1 min, and 72 °C 4 min; final
extension at 72 °C for 10 min. The PCR products were directly
sequenced by using an automatic sequencer. To prepare the total RNA,
S. solfataricus P2 strain cells were grown at 0.6 A600 (midexponential phase) in the
indicated culture medium; cells were lysated by three cycles of
freeze-thawing (2 min at
Primer extension experiments were performed as reported previously
(23); the synthetic oligonucleotides (Genenco, Florence, Italy)
used are as follows: Fext1, 5'-CACTTTTCTTAAAGACCTTAG-3'; Fext2,
5'-GACTATATACTTTTTTAATGAAC-3'.
Plasmids Preparation--
The SSO3060 ORF, encoding for the
C-terminal fragment of the
The DNA fragment containing SSO11867 plus SSO3060 was cloned by PCR by
using the standard program, the synthetic oligonucleotide Site-directed Mutagenesis--
The vector pGEX-11867/3060,
described above, was used as template for site-directed mutagenesis
experiments (24). The synthetic oligonucleotides used were Protein Purification--
E. coli BL21(DE3)/pET-3060
was grown in 2 liters of Super Broth at 37 °C. Gene expression was
induced by the addition of 1 mM
isopropyl-1-thio-
Growth of E. coli BL21(RB791)/pGEX-11867/3060 and total
proteins extraction was performed as described for pET-3060. The crude extract was applied to a glutathione-Sepharose 4B column (Amersham Biosciences) that had been equilibrated with the same buffer. After 10 column volumes washing with PBS buffer (without Triton), the fusion
protein was eluted from the column by the addition of 500 mM Tris-HCl, pH 8.0, supplemented with 10 mM
reduced glutathione, at room temperature (22-25 °C); the eluate is
collected in 1.5-ml volume fractions and assayed for GST activity at
25 °C. Active fractions were collected and stored at 4 °C. The
active pool was then subjected to thrombin treatment; to this aim,
pooled fractions (about 30 ml) were incubated at 4 °C overnight with
30 units of thrombin solution (Amersham Biosciences). This sample,
analyzed by SDS-PAGE, did not revealed any band compatible with the
molecular weight of the full-length
Mutant Enzyme Characterization--
The standard assay of
Thermal activity of Ss
Molecular mass of denatured Ss Enzymatic Synthesis of Fucosides--
Thin layer chromatography
and NMR spectra were performed as reported previously (27). The pNp-Fuc
(0.17 mmol) and p-nitrophenyl-
For
For
During pNp-Fuc hydrolysis reactions, the formation of a disaccharidic
component was noticed along with fucose. Purification, acetylation, and
NMR characterization of the disaccharidic compound as reported above
revealed it to be
For
Identification and Sequence Analysis of the
Experiments of primer extension on the same total RNA preparations were
performed with oligonucleotides that anneal on the SSO11867 and SSO3060
ORFs (Fig. 4). The primer Fext2 showed a specific transcriptional initiation site nine nucleotides upstream from
the first ATG of the SSO11867 ORF (Fig. 4); this was confirmed by
primer Fext1, which, however, showed additional initiation sites (Fig.
4). The strongest signal showed by Fext1 maps on the third base of the
first putative ATG of the C-terminal ORF SSO3060 (Fig. 4). However, the
expression of the ORF SSO3060 in E. coli as pET3060 did not
produce any protein band of the expected molecular weight (46.5 kDa),
and no detectable
Potential promoter sequences were found at Preparation of a Full-length
To test whether a functional
The obtained pGEX-frameFuc plasmid was used to express the mutant
enzyme in E. coli. An intense protein band of molecular weight compatible with the GST- Characterization of the
The residual activity of Ss
The enzyme followed the classical Michaelis-Menten behavior on pNp-Fuc
at 65 °C, with Km and kcat
values of 0.028 ± 0.004 mM and 287 ± 11 s Ss
The analysis by TLC of the hydrolysis reaction mixture containing only
pNp-Fuc revealed the presence of fucose, of pNp, and of a compound
that, after acetylation and NMR spectroscopy, was identified as
Among the disrupted genes in S. solfataricus, we have
identified two ORFs, SSO11867 and SSO3060, homologous to eukaryal and bacterial Two mutations, in key positions to mimic programmed The substrate characterization revealed that Ss In retaining enzymes, when acceptors different from water
intercept the reactive glycosyl-enzyme intermediate, they promote transglycosylation reactions (Fig. 9).
The ability of Ss1 frameshift and encoding for the N- and the
C-terminal fragments, respectively, of an
-L-fucosidase.
We report here that these ORFs are actively transcribed in
vivo, and we confirm the presence of the
1 frameshift between
them at the cDNA level, explaining why we could not find
-fucosidase activity in S. solfataricus extracts.
Detailed analysis of the region of overlap between the two ORFs
revealed the presence of the consensus sequence for a programmed
1
frameshifting. Two specific mutations, mimicking this regulative
frameshifting event, allow the expression, in Escherichia
coli, of a fully active thermophilic and thermostable
-L-fucosidase (EC 3.2.1.51) with micromolar substrate
specificity and showing transfucosylating activity. The analysis of the
fucosylated products of this enzyme allows, for the first time,
assigning a retaining reaction mechanism to family 29 of
glycosyl hydrolases. The presence of an
-fucosidase putatively
regulated by programmed
1 frameshifting is intriguing both with
respect to the regulation of gene expression and, in post-genomic era,
for the definition of gene function in Archaea.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-L-fucose is an important
constituent of the carbohydrate chains of glycoconjugates involved in a
variety of biological events as growth regulators and receptors in
signal transduction, cell-cell interactions, and antigenic response
(1).
-L-fucosylated oligosaccharides derived from
xyloglucan, a plant cell wall component that controls cell expansion, have been shown to regulate auxin- and acid pH-induced growth (2). In
mammals, oligosaccharides containing fucose have been found, for
instance, in human milk and in blood group substances (3), and they are
reported to play important roles in fertilization (4) and in adhesion
processes of viruses, bacteria, and other parasites (5). Changes in
fucosylation patterns have been observed in several physiological
events including pregnancy (6), programmed cell death of different cell
types (7), and in a variety of pathological events including diabetes
(8) and colon and liver carcinomas (9, 10). In addition, the
determination of
-fucosidase activity can be used to predict the
development of colorectal, ovarian, and hepatocellular carcinomas
(11-13), whereas the deficiency in this enzyme causes fucosidosis, a
well known lysosomal storage disorder (14). The central role of fucose
derivatives in biological events explains the interest in
-L-fucosidase and fucosyltransferase activities.
-fucosidases (EC 3.2.1.51) from a variety of sources, including human and several pathogenic bacteria (15). No structural data are
available about this class of enzymes, and the residues involved in
catalysis are still unknown. Recently, it has been shown (16) that the
-fucosidase from Thermus sp. Y5 performs the hydrolytic reaction with the retention of the anomeric configuration. The analysis
of the genome of the hyperthermophilic Archaeon Sulfolobus solfataricus (17) revealed the presence of two ORFs, annotated as
SSO11867 and SSO3060, separated by a
1 frameshift, that are homologous to the N- and the C-terminal fragments, respectively, of
full-length bacterial and eukaryal GH29 fucosidases.
1 frameshifts are by far the most prevalent (19); they
have been well characterized in RNA viruses, but the basic molecular
mechanisms governing these events are almost identical from yeast to
humans (20). In Archaea, documented recoding events are limited to
readthrough (21); no proofs of programmed
1 frameshifting have been
reported in this domain so far.
-fucosidase, we have cloned and expressed these genes in
Escherichia coli. Two specific mutations, designed on the
basis of the programmed
1 frameshifting mechanism, allow the
expression of a full-length thermophilic and thermostable
-L-fucosidase, with micromolar substrate specificity,
which promotes transfucosylation reactions by following a
retaining reaction mechanism. This is the first evidence of
an
-fucosidase from Archaea, and our data unequivocally demonstrate
that GH29 enzymes follow a retaining reaction mechanism.
Furthermore, the data presented give support to the hypothesis that
translational recoding could be used to regulate gene expression in Archaea.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Fucosidase Expression Analysis in S. solfataricus--
Genomic DNA from S. solfataricus MT4 and
P2 strains was prepared by the NucleoSpin Tissue kit (Mackerey-Nagel,
Germany). The DNA fragment of 833 nucleotides (positions 1-833),
containing the region overlapping the ORFs SSO11867 and SSO3060, was
prepared by PCR, by using the following synthetic oligonucleotides
(Genenco, Florence, Italy):
-fuc-3,
5'-GAGGAAGATCTATGTCACAAAATTCTTACAAAATC-3';
-fuc-833,
5'-TTGCTTGTAAATTATTACGGG-3'.
70 °C; 2 min 37 °C), and total RNA was
extracted by the RNeasy Kit (Qiagen, Germany). Contaminating DNA was
eliminated by digestion with DNase I RNase-free (Promega). Reverse
transcriptase (RT)-PCR was performed by using the Titan One Tube RT-PCR
system (Roche Molecular Biochemicals) and the same oligonucleotides
shown above. The PCR program used is as follows: cDNA synthesis and
pre-denaturation for 31 min at 50 °C and 2 min at 94 °C;
amplification by 40 cycles of 15 s at 94 °C, 30 s at
45 °C, and 2 min at 72 °C; final extension of 10 min at 72 °C.
The cDNA products obtained were directly sequenced as described
above, with no further purification steps.
-Fucosidase activity was searched in extracts of S. solfataricus grown in different culture media (minimal salts
medium supplemented with yeast extract (0.1%) or casamino acids,
glucose, fucose, or sucrose (each at 0.1%), and different combinations
of these). Enzymatic assays in S. solfataricus extracts were
performed by using up to about 0.65 mg of crude extracts in standard
conditions (see below for the standard enzymatic assay) on
p-nitrophenyl-
-L-fucopyranoside (pNp-Fuc) at
75 °C for up to 5 h; at these conditions the hydrolysis of the
substrate was identical to that of the blank mixture without protein.
-fucosidase, was cloned by amplification
of S. solfataricus, strain P2, chromosomal DNA via PCR by
using the standard program (see above), and the following
synthetic oligonucleotides (Genenco, Florence, Italy):
-fuc-1,
5'-GGGAATTCATATGTTCACTGGAGAGAATTGGGAACCGTA-3';
-fuc-2,
5'-CGCGGATCCCTATCTATAATCTAGGATAACCC-3' which introduce an
NdeI and BamHI sites at the 5', just before the
first ATG, and at the 3' ends of the ORF, respectively. The resulting
DNA fragment was cloned in the pET29a plasmid (Novagen), obtaining the
vector pET-3060, in which SSO3060 ORF is under the control of the
isopropyl-1-thio-
-D-galactopyranoside-inducible T7 RNA polymerase promoter that drives high expression levels in bacterial hosts. The ORF obtained after amplification was controlled by DNA sequencing.
-fuc-3 described above, and
-fuc-4,
5'-GAGGAAGATCTCTATCTATAATCTAGGATAACCC-3'. Both oligonucleotides
introduce BglII sites at the 5', just before the first ATG
of the SSO11867 ORF, and at the 3' end of the SSO3060 ORF. The
resulting DNA fragment was cloned in the pGEX-2TK plasmid (Amersham
Biosciences). In the plasmid obtained, pGEX-11867/3060, GST was fused
to the N-terminal of the SSO11867 gene product; the fusion and the
entire DNA fragment obtained after amplification were controlled by DNA sequencing.
-fuc-3
and
-fuc-4, and the following mutagenic oligonucleotide
FrameFuc,
5'-phosphate-GTTACTGGGCCGAAATTCTTTAGGTGATATTGG-3' where the mismatched nucleotides are underlined. The DNA fragment containing the mutations was subcloned in the vector pGEX-11867/3060; the mutant clone, named pGEX-frameFuc, was identified by direct sequencing and was completely re-sequenced.
-D-galactopyranoside when the culture
reached an A600 of 1.0. Growth was allowed to
proceed for 16 h, and cells were harvested by centrifugation at
5,000 × g and frozen at
20 °C. The resulting cell
pellet was thawed, resuspended in 2 ml g
1 cells of 50 mM sodium phosphate buffer, pH 7.4, 150 mM
NaCl, 1% (v/v) Triton X-100 (PBS-Triton buffer), and homogenized by treatment with a cell disruption equipment (Constant Systems Ltd., Warwick, UK). After disruption, the homogenate was diluted 1:1 with the
same buffer and centrifuged for 30 min at 30,000 × g; cell debris was discarded, and the crude extract was diluted 10-fold in
the same buffer; no activity on pNp-Fuc at 65 °C was observed (see
below for the standard enzymatic assay).
-fucosidase but showed low
activity on pNp-Fuc at 65 °C.
-fucosidase was expressed from E. coli
BL21(RB791)/pGEX-frameFuc and extracted as described for pET-3060. The
GST binding was performed by adding 3 ml of the glutathione-Sepharose 4B matrix (Amersham Biosciences), equilibrated with the same buffer, to
the crude extract and incubated overnight at 4 °C. After the binding, the matrix was packed, and after 30 column volumes washing with PBS buffer (without Triton), the matrix was resuspended in 1 volume of PBS buffer and incubated overnight at 4 °C with 60 units
of thrombin solution. The efficiency of thrombin cleavage was tested by
loading onto SDS-PAGE an amount of the matrix slurry before and
after the thrombin treatment. Thereafter, the soluble and GST-free
-fucosidase protein was recovered by 5 column volumes washes with
PBS buffer. Washes containing the
-fucosidase protein were pooled
and concentrated by ultrafiltration on an Amicon YM30 membrane (cut-off
30,000 Da). After this treatment, the
-fucosidase was >95% pure by
SDS-PAGE and was used for all the subsequent characterizations. The
purification procedure yielded about 2 mg of pure protein from
13.7 g of wet cell pellet. The sample stored at 4 °C is stable
for several months. Direct sequencing of the N-terminal of the purified
enzyme produced the sequence: Ser-Val-Gly-Ser-Met-Ser-Gln-Asn-Ser-Tyr-Lys-Ile-Leu-Lys-,
in which the underlined amino acids correspond to the N terminus of SSO11867.
-fucosidase activity was performed at 65 °C in 50 mM
sodium phosphate buffer at pH 6.3, with pNp-Fuc substrate at the final
concentration of 1 mM. The molar extinction coefficient of
p-nitrophenol is 9340 M
1
cm
1 measured at 405 nm, at 65 °C, in 50 mM
sodium phosphate buffer, pH 6.5. One unit of enzyme activity was
defined as the amount of enzyme catalyzing the hydrolysis of 1 µmol
of substrate in 1 min at the conditions described. Spontaneous
hydrolysis of the substrate (about 0.1%) was subtracted by using
appropriate blank mixtures without enzyme. The kinetic constants of the
-fucosidase mutant (Ss
-fuc) on pNp-Fuc were measured at 65 °C
in 50 mM sodium phosphate buffer, pH 6.3, by using
substrate concentrations in the range 0.005-3 mM. The
protein concentration in the reaction mixture was 0.4 µg
ml
1. All kinetic data were calculated as the average of
at least two experiments and were plotted and refined with the program GraFit (25).
-fuc was analyzed by assaying the enzyme (0.8 µg) on pNp-Fuc substrate concentrations of 1 and 3 mM in
the temperature ranges of 40-65 and 70-95 °C, respectively. Thermal stability was tested by incubating pure enzyme (0.08 mg ml
1) in PBS buffer at the indicated temperatures, as
reported previously (26).
-fuc was determined by SDS-PAGE in
both reducing and non-reducing conditions. Molecular mass of native
Ss
-fuc was determined by gel filtration on a Superose 6 column 26/60
HiLoad (Amersham Biosciences) runs in PBS buffer at a 0.3 ml
min
1 flow rate; molecular weight markers were run under
the same conditions.
-D-glucoside (pNp-Glc) (0.51 mmol) were dissolved in 3 ml of phosphate buffer 20 mM, pH 7.0; 200 µl of enzyme preparation (0.08 mg of
total protein) were added, and the reaction was started at 75 °C,
under agitation, in a sealed vial for 5 h up to total consumption
of donor as monitored by TLC (CHCl3/MeOH/H2O,
65:25:4, v/v). The total reaction mixture was cooled, and the product
was purified by RP-18 column chromatography; the fractions containing
fucosylated products were purified by preparative TLC. After
acetylation, the following compounds were obtained in 14% total yield
with respect to the donor.
-L-Fuc-(1-3)-
-D-Glc-O-pNp,
COSY and 1H-13C NMR correlations permit the
assignment of 1H and (13C signals) of
carbohydrate moiety as follows. Glc moiety: 5.74 (94.2) H1, 5.09 (72.2)
H2, 4.37 (74.1) H3, 5.19 (69.1), H4, 3.89 (69.16) H5, 4.13-4.03 (61.6)
H6a, H6b;
-L-Fuc moiety: 5.46 (96.5) H1, 5.06 (68.6)H2,
5.24 (67.2) H3, 5.27 (70.8), H4, 4.12 (65.3) H5, 1.13 (15.9) H6.
[
]
-L-Fuc-(1-2)-
-D-Glc-O-pNp,
COSY and 1H-13C NMR correlations permit the
assignment of 1H and (13C signals) of
carbohydrate moiety as follows. Glc moiety: 5.77 (95.6) H1, 3.95 (76.7)
H2, 5.67 (70.9) H3, 5.11 (70.6), H4, 3.99 (68.5) H5, 4.03-4.24 (61.3)
H6a, H6b.
-L-Fucp moiety: 5.24 (98.3) H1,
5.08 H2, 5.17 H3, 5.19 H4 (67.9,67.4, 70.4 overlapping correlation), 4.05 (65.5) H5, 0.71 (15.6) H6. [
]
-L-Fuc-(1-3)-
-L-Fuc-O-pNp.
-L-Fuc-(1-3)-
-L-Fuc-O-pNp,
1H (COSY) of disaccharidic moiety: 5.90 (H-1 aryl-linked
fucose), 5.32 (H-2 aryl-linked fucose), 5.30 (H-4 aryl-linked fucose),
5.27 (2H, H3-H4 external fucose unit), 5.22 (H2 external fucose)
5.20 (H-1 external fucose), 4.45 (H-3 aryl-linked fucose), 4.41 (H-5
aryl-linked fucose) 4.06 (H-5 external fucose) 1.21 (H-6 aryl-linked
fucose) 1.11 (H-6 external fucose). 13C NMR signals: 94.8, 93.8, 71.0, 69.5, 69.1, 69.0, 67.7, 67.0, 66.4, 64.8, 15.9, 16.0.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Fucosidase
Locus--
S. solfataricus, strain P2, is a
hyperthermophilic Archaeon able to grow at acidic pH (pH 3-5) and at
high temperatures (80-87 °C). In an effort to determine the full
set of glycosyl hydrolases produced by this Archaeon, we analyzed the
ORFs putatively encoding for these enzymes in the sequenced genome
(17). Two of these ORFs, SSO11867 and SSO3060, encode for 81 and 426 amino acids polypeptides, respectively, and are homologous to the N-
and C-terminal parts, respectively, of GH29
-fucosidases (Fig.
1) (15). SSO11867 and SSO3060 are
separated by a
1 frameshift in a 40-base overlap (Fig.
2); this is consistent with the
observation that no
-fucosidase activity could be found in S. solfataricus extracts obtained from cells grown on different media
(glucose, fucose, or sucrose as the only energy sources or in
combination with yeast extract and casamino acids). To test if the
frameshift was due merely to sequencing errors, DNA fragments
containing the overlapping region were amplified from the genome of
S. solfataricus P2 and MT4 strains, which are strictly
taxonomically related (29) and directly sequenced. The sequences were
identical to the one published (Fig.
3A), confirming the presence
of a
1 frameshift between these two ORFs. Moreover, a cDNA
fragment was amplified by RT-PCR from a total RNA preparation of
S. solfataricus, strain P2, and directly sequenced (Fig.
3B). Again, the obtained sequence turned out to be identical
to that available from the data bank with no ambiguities (Fig.
3C), indicating that the population of RNA amplified by
RT-PCR was identical to the genomic DNA; this result excludes the
possibility of RNA-editing events and demonstrates that SSO11867 and
SSO3060 are co-transcribed in vivo.
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Fig. 1.
Alignment of SSO11867 with non-redundant
amino acid sequences of GH29
-fucosidases. Sequences were aligned with the
program MultAlin (28). GenBankTM accession numbers are in
parentheses. Celeg, Caenorhabditis
elegans (P49713); Cfam1-2, Canis familiaris
(P48300 and U29765, respectively); Hsap1-3, Homo
sapiens (Q14334, M10355, and A1031320, respectively);
Mmus1-2, Mus musculus (AK002230 and AK008653,
respectively); Rnor, Rattus norvegicus (P17164);
Hror, Halocynthia roretzi (AB070600);
Dmel, Drosophila melanogaster (AAF50054.1);
Ddisc, Dictyostelium discoideum (P10901);
Tmar, Thermotoga maritima (Q9WYE2);
Ccresc, Caulobacter crescentus (AAK22780);
Xaxo, Xanthomonas axonopodis (AAM37917.1);
Xfast1-2, Xylella fastidiosa (AAF85750,
AAF82919, respectively); Micr1-2, Microscilla
sp. PRE1 (AAK62841.1 and AAK62842.1, respectively); Cperf1-3,
Clostridium perfringens (Q8XNK9, Q8XMM5, and Q8XJ85,
respectively); Scoel, Streptomyces coelicolor
(AAD10477); Strept, Streptomyces sp. 142 (AAD10477). The last amino acids of the SSO11867 ORF, encoded in the
zero frame of the region of overlap between the SSO11867 and SSO3060
ORFs, are underlined.
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Fig. 2.
The -fucosidase
locus in S. solfataricus. The N-terminal
SSO11867 ORF is in the zero frame, and the C-terminal SSO3060 ORF, for
which only a fragment is shown, is in the
1 frame. The slippery
heptameric sequence is underlined; the rare codon is
boxed, and the arrows indicate the stem of the
putative mRNA secondary structure. The amino acids involved in the
programmed
1 frameshift and the first codon translated after this
event in the
1 frame are shaded.
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Fig. 3.
Sequence analysis of the
-fucosidase locus in S. solfataricus. A, electropherogram of the DNA
sequence of the PCR products obtained from S. solfataricus
genome, strain P2; the region of overlap between the two ORFs SSO11867
and SSO3060 is underlined. B, agarose gel showing
the products of RT-PCR from total cellular RNA; lane 1,
control (amplification of total RNA without reverse transcriptase
enzyme); lane 2,
-fucosidase; lane 3, XylS
(positive control); lane 4, molecular weight markers, 100-bp
DNA ladder. C, electropherogram of the DNA sequence of the
RT-PCR product shown in B, lane 2; the region of
overlap between the two ORFs SSO11867 and SSO3060 is
underlined.
-fucosidase activity on the pNp-Fuc substrate at
65 °C was found. This suggests that this ORF could not express a
functional enzyme independently; this is not surprising because in the
N-terminal ORF SSO11867 an amino acidic sequence conserved among GH29
-fucosidases can be found (Fig. 1).
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Fig. 4.
Analysis of transcriptional initiation sites
in SSO11867 and SSO3060 ORFs by primer extension. Top,
primer extension: lanes 1 and 2, primer extension
controls (without total RNA) of Fext1 and Fext2 oligonucleotides,
respectively; lanes 3 and 4, primer extension of
Fext1 and Fext2 oligonucleotides, respectively. The signals
corresponding to the initiation sites upstream from SSO11867
(site A) and SSO3060 (site B) are indicated by
closed arrows. The asterisks indicate an
aspecific signal of Fetx1 observed also in the control.
Bottom, -fucosidase locus in S. solfataricus:
the TATA box and the TFB-responsive element (BRE) are
boxed; the first ATGs of SSO11867 and SSO3060 are
underlined with a dark line; the slippery
heptameric sequence is underlined; the amino acids involved
in the programmed
1 frameshift are shaded. The initiation
sites A and B are shown with closed arrows. The sequences
corresponding to Fext1 and Fext2 oligonucleotides, mapping in the
C-terminal SSO3060 and N-terminal SSO11867 ORFs, respectively, are
underlined with open arrows.
26 nucleotides from the
transcription initiation site upstream from SSO11867, whereas no such
consensus could be identified in the region upstream from SSO3060 (Fig.
4).
-L-Fucosidase--
The
complete S. solfataricus
-fucosidase locus, with SSO11867
and SSO3060 in different reading frames (pGEX-11867/3060), drives the
expression in E. coli of trace amounts of
-fucosidase activity; after removal of GST a specific activity of 2.3 × 10
2 units mg
1 on pNp-Fuc at 65 °C was
found. A detailed analysis of the DNA sequence of the region of overlap
between the two ORFs revealed the presence of a stretch of six adenines
followed by a thimine (Fig. 2) that resembles one of the heptamers that
are involved in programmed
1 frameshifting (19). Typically, the sites
cis-regulating these events consist of a "slippery"
heptameric sequence of the general form X-XXY-YYZ (codons are in the
zero frame, and X, Y, and Z can be identical or different nucleotides)
and often include an upstream Shine-Dalgarno sequence and a downstream
mRNA secondary structure. In fact, it has been reported that a
Shine-Dalgarno sequence along the mRNA and pseudoknots or
stem-loops promote the pausing of the ribosome (19). The sequence of
overlap between the ORFs SSO11867 and SSO3060 presents a similar
organization. The slippery sequence A-AAA-AAT was immediately followed
by a stem loop, and the rare codon CAC, which is used at low frequency by S. solfataricus (4.7 per thousand in the S. solfataricus genome), was found upstream from the slippery
sequence (Fig. 2). This rare codon presumably plays the function of the
Shine-Dalgarno sequence, which is rarely observed in isolated genes in
this Archaeon (30), by inducing the pausing of the ribosome and
increasing the frequency of the frameshifting event. These observations
raised the hypothesis that a programmed
1 frameshift could promote
the expression of an active enzyme in S. solfataricus;
however, testing this hypothesis in vivo is impaired by the
lack of molecular genetic tools for hyperthermophilic Archaea.
-fucosidase could be produced, a single
frame between the ORFs was restored by site-directed mutagenesis. In
the mechanism of programmed
1 frameshifting proposed for Eukarya and
bacteria, two tRNAs, hybridized to the XXY and YYZ codons of the
X-XXY-YYZ sequence, are proposed to slip simultaneously backwards on
the mRNA to the
1 frame, hybridizing to XXX and YYY codons. The
AAT triplet, coding for Asn-78 in SSO11867, corresponds to the YYZ
codon, and is the last one decoded in the zero frame (19); after this
triplet, the ribosome would shift onto the TTC codon of the Phe-10
(SSO3060 numbering) continuing the translation in the
1 frame (Fig.
2). To obtain the fused gene, we performed site-directed mutagenesis in
the pGEX-11867/3060 vector in which glutathione
S-transferase (GST) enzyme was fused to the N-terminal of
SSO11867. On the basis of the mechanism proposed, a T in the region
following the slippery heptamer was introduced; moreover, we introduced
the conservative mutation AAA
AAG (encoding for Lys-77 in SSO11867)
to increase the translational fidelity by disrupting the heptameric
sequence. These mutations changed the putative
1 frameshifting site
from CTA-AAA-AAT-TCG-GCC (zero frame, slippery heptamer
underlined) to
CTA-AAG-AAT-TTC-GGC (the
nucleotides in boldface were originally in the
1 frame; the mutations
are underlined). As a result, the ORFs are in the same translation
frame producing a single polypeptide.
-fucosidase fusion (84.4 kDa) was
observed after affinity chromatography of the crude extracts on
glutathione-Sepharose 4B (Fig. 5); the
removal of GST by thrombin cleavage was performed directly onto the
column producing, by a single purification step, a protein more than
95% pure (Fig. 5). The recombinant enzyme showed the expected 57-kDa
molecular mass (corresponding to the predicted full-length
polypeptide of 495 amino acids) and revealed a specific activity of
32.1 units mg
1 on pNp-Fuc at 65 °C; it was termed
Ss
-fuc.
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Fig. 5.
SDS-PAGE analysis of expressed
-fucosidase. Lane 1, slurry of
glutathione-Sepharose 4B matrix with bound GST-
-fucosidase fusion
expressed from E. coli BL21(RB791)/pGEX-frameFuc, before the
thrombin cleavage; lanes 2 and 3, same as
lane 1, first and second elution, respectively, after
thrombin treatment; lanes 4 and 5, purified
Ss
-fuc 2 and 4 µg, respectively. The molecular weight markers used
are as follows: phosphorylase b (97,000), bovine serum
albumin (66,000), ovalbumin (45,000), and carbonic anhydrase
(30,000).
-Fucosidase--
The native molecular
weight of the enzyme, expressed and purified as described above, was
analyzed by gel filtration under native conditions: a single peak
containing thermophilic
-fucosidase activity eluted between
thyroglobulin (660 kDa) and ferritin (490 kDa) for a molecular mass of
about 508 ± 22 kDa, suggesting that Ss
-fuc could be a decamer
in solution (Fig. 6). The enzyme has a
broad pH dependence on pNp-Fuc at 65 °C, showing almost the same
specific activity in the range pH 3.3-6.3 on different buffer systems
(data not shown). The thermal activity of Ss
-fuc is reported in Fig.
7A; the activity on pNp-Fuc
increased sharply up to the optimal temperature of 95 °C, the
highest temperature tested. The calculated value of activation energy
(Ea) for the hydrolytic reaction of this substrate
was 91 ± 2 kJ mol
1.
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Fig. 6.
Gel filtration analysis of
Ss -fuc. Elution profile at 280 nm from a
Superose 6 column. The elution volumes are as follows: 7.39 ml of blue
dextran; 12.09 ml of thyroglobulin (669,000); 13.53 ml of Ss
-fuc;
14.03 ml of ferritin (490,000); 15.48 ml of aldolase (158,000); 16.01 ml of bovine serum albumin (67,000); 18.46 ml of ribonuclease A
(13,700).
View larger version (16K):
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Fig. 7.
Activity and stability of
Ss -fuc. A, thermal activity
and derived Arrhenius plot (inset) of Ss
-fuc on pNp-Fuc.
B, thermal stability of Ss
-fuc at 75 (open
circles), 80 (closed circles), 85 (open
squares), 90 (closed squares), and 95 °C (open
triangles). The Arrhenius plot derived from the data in the
temperature range 80-95 °C is reported in the
inset.
-fuc after preincubation at temperatures
ranging from 75 to 95 °C was followed for up to 2 h on pNp-Fuc
at 65 °C (Fig. 7B); the enzyme displayed high stability at 75 °C, showing even a 40% activation after 30 min of incubation and maintaining 60% residual activity after 2 h at 80 °C. The calculated activation energy for the inactivation reaction, calculated from the Arrhenius plot shown in the inset of Fig.
7B, turned out to be 1120 ± 167 kJ mol
1,
more than 10-fold the Ea measured for the catalyzed reaction, confirming the extreme thermal stability of
Ss
-fuc.
1, respectively, which led to an extremely high
kcat/Km (10,250 s
1 mM
1). Ss
-Fuc shows high
selectivity for the pNp-Fuc substrate; in fact, even after extensive
incubation (more than 2 h) at the optimal conditions, no activity
was observed on aryl substrates such as pNp-
-L-arabinoside, pNp-
-D-galactoside,
pNp-
-D-glucoside, pNp-
-D-mannoside, pNp-
-D-xyloside, and pNp-
-L-rhamnoside.
No activity was observed on the sulfated polysaccharide fucoidan from
Fucus vesiculosus, formed by a repetition of
-(1,3)- and
-(1,4)-fucose residues, and on its products obtained by acetolysis
(31) and deprotected by alkaline methanolysis.
-fuc Promotes Transfucosylation Reactions--
In order to
test the transfucosylating activity of Ss
-fuc, we investigated the
pyranosidic acceptor pNp-Glc. In the reaction in which pNp-Fuc and
pNp-Glc were the donor and the acceptor substrates, respectively, in
5 h Ss
-fuc cleaved the donor, forming fucosylated products with
14% total yield with respect to pNp-Fuc. Interglycosidic linkages were
determined by NMR spectroscopy (COSY and 1H-13C
NMR correlation), and the signals were assigned as reported under
"Experimental Procedures." In the COSY spectrum of one of the
products, following the correlations through pyranosidic protons of
-D-Glc unit and starting from the anomeric signal at
5.74 ppm (J = 3.9 Hz), it is easy to detect H3 proton, which
correlates with carbon signal at 74.1 ppm (glycosylation shifts),
identifying this compound as
-L-Fucp-(1-3)-
-D-Glc-O-pNp.
Using the same reasoning, the interglycosidic linkages of the other
disaccharide (
-1-2, 3.95/76.7) were easily assigned to
-L-Fucp-(1-2)-
-D-Glc-O-pNp.
-L-Fuc-(1-3)-
-L-Fuc-O-pNp.
This disaccharide was used as substrate for enzymatic hydrolysis;
interestingly, this reaction mixture revealed, by TLC analysis, the
presence of fucose and pNp-Fuc products after incubation at 65 °C
for 3 min and the complete hydrolysis of the substrate after 10 min
(Fig. 8). This indicated that the enzyme
cleaved the aryl disaccharide starting from its non-reducing end.
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Fig. 8.
TLC analysis of the hydrolysis of
-L-Fuc-(1-3)-
-L-Fuc-O-pNp.
T0, starting reaction time;
T3, T5, and
T10, 3-, 5-, and 10-min reaction time,
respectively. Fuc,
-L-fucose;
pNpFucFuc,
-L-Fuc-(1-3)-
-L-Fuc-O-pNp.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-fucosidases from family 29 of glycosyl hydrolases; the
presence of a
1 shift between these ORFs was confirmed, and we found
that they are actively co-transcribed in S. solfataricus. A
complete programmed
1 frameshifting regulation site has been identified, suggesting that SSO11867 and SSO3060 could be expressed in vivo by following this mechanism. Remarkably, the two
ORFs express in E. coli trace amounts of
-fucosidase
activity, confirming that the frameshifting cassette promotes
non-regulated frameshifting during overexpression of foreign genes in
E. coli (19). Primer extension analysis showed two major
transcriptional initiation sites: the first mapped upstream from the
two ORFs, and the second started from the G of the first ATG of the
SSO3060 ORF. This ORF could not express a functional protein with
-fucosidase activity in E. coli, suggesting that
post-transcriptional cleavage may occur on the longer transcript.
However, the possibility that SSO3060 expresses in S. solfataricus a polypeptide with a different function, thus
explaining the programmed
1 frameshifting regulation, cannot be ruled
out; experiments are in progress to study the expression in
vivo of these genes.
1 frameshifting,
made it possible to drive the expression of the full-length
-fucosidase Ss
-fuc that is optimally active at 95 °C.
Remarkably, the enzyme is stable at 80 °C, the temperature at which
S. solfataricus optimally grows; these observations are
strong evidences that the enzyme produced by following the programmed
1 frameshifting could be stable and active in vivo. These
results indicate that SSO11867 and SSO3060 ORFs encode for the first
archaeal
-fucosidase identified so far, suggesting that they may not
be pseudogenes and that programmed
1 frameshifting could be present
in this living domain.
-fuc catalyzes the
hydrolysis of pNp-Fuc with high efficiency and specificity; in the
framework of our mechanistic studies on thermophilic glycosyl hydrolases, the reaction mechanism of Ss
-fuc was analyzed in detail.
Glycosyl hydrolases follow two distinct mechanisms that are termed
retaining or inverting if the enzymatic cleavage
of the glycosidic bond liberates a product with the same or the
opposite anomeric configuration of the substrate, respectively (32). Both classes of enzymes employ a pair of carboxylic acid residues in
catalysis and operate via transition states with substantial oxocarbenium ion character. The alignment of the amino acid sequences of the 24 GH29
-fucosidases shown in Fig. 1 revealed two residues of
aspartic/glutamic acid that are completely conserved in this family
(Asp-124 and Asp-146 in Ss
-fuc numbering) and are the best
candidates to be involved in catalysis as the nucleophile or the
acid/base of the reaction (not shown); experiments are currently in
progress to test the functional role of these residues.
-fuc to function in the transglycosylation mode
allowed us to demonstrate experimentally that the enzyme catalyzes the
formation of the
-(1,2) and
-(1,3) bonds between fucose and the
glucose of pNp-Glc. The
-anomeric configuration of the
interglycosidic linkages in the products unequivocally indicates that
GH29
-fucosidases follow a retaining reaction mechanism;
this is the first time that the mechanism followed by glycosyl
hydrolases from this family has been experimentally demonstrated by
following transglycosylation reaction. Moreover, the activity observed
on
-L-Fuc-(1-3)-
-L-Fuc-O-pNp revealed that Ss
-fuc is an exo-glycosyl hydrolase that attacks the
substrates from their non-reducing end. The disaccharide
-L-Fucp-(1-3)-
-D-Glc-O-pNp was synthesized previously (33) by using a mesophilic
-L-fucosidase in a reaction using the fluoroderivative
of the donor and glucose as acceptor in 34% yield. In our case no
efforts were made for the optimization of the reaction conditions
(keeping as low as possible the acceptor/donor equivalent ratio useful
for synthetic application with rare acceptors). However, the
thermophilic nature of Ss
-fuc is of interest for biotechnological
exploitation of its transferring capability (34).
View larger version (15K):
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Fig. 9.
Proposed retaining
reaction mechanism of Ss -fuc.
Family 29 of glycosyl hydrolases includes enzymes from plants,
vertebrates, and pathogenic microbes of plants and humans. The data
presented here demonstrate that this enzymatic activity is present in
all the three living domains. The ORF TM0306 from the bacterium
Thermotoga maritima, putatively encoding for an -fucosidase, is the only other known example from hyperthermophiles (35); recently, an
-L-fucosidase secreted by the
moderate thermophile Thermus sp. Y5 has been isolated and
characterized (16). Interestingly, Ss
-fuc showed the highest amino
acid sequence identity (40%) with a putative
-fucosidase from the
phytopathogen bacterium Xanthomonas (36), whereas only 25%
identity with the T. maritima enzyme was observed; this is
surprising because higher similarity between enzymes from
hyperthermophiles would be expected. Structural comparisons of
Ss
-fuc with Thermus sp. enzyme, which is a secreted tetrameric protein, are hampered by the only partial sequence available
for the eubacterial enzyme; however, Ss
-fuc did not show signal
peptides for secretion, and no
-fucosidase activity was found in
S. solfataricus media.
-Fucosidases in higher plants and in mammals are associated with
different mechanisms of cell growth and regulation, because they are
involved in the modification of fucosylated glucans (1). By contrast,
at present, there are only limited data on
-fucosidases from
eukaryal and bacterial microorganisms. The presence, in S. solfataricus, of glycosylated proteins has been reported recently (37); although the composition of their carbohydrate moiety is unknown,
different enzymatic activities for the synthesis, modification, and
hydrolysis of these glycosidic bonds should be present in this Archaeon
and regulated in some manner. The activity of Ss
-fuc (efficient
hydrolysis of aryl fucosides, very high substrate specificity,
transfucosylation capability) may suggest its involvement in
vivo in these biological processes.
Relatively little is known about the function of the 22 glycosyl
hydrolases of S. solfataricus; several genes encoding for this class of enzymes, including SSO11867 and SSO3060, an
-glucosidase (SSO3051), a
-glucuronidase (SSO3036), a
-xylosidase (SSO3032), and the clustered
-xylosidase (XylS), and
-glycosidase (Ss
-gly) (SSO3022 and SSO3019, respectively) map in
the same region of about 50 kb and are likely to be involved in the
degradation of sugars for energy metabolism. In particular, we reported
previously that XylS and Ss
-gly hydrolyze xyloglucan
oligosaccharides in vitro in a cooperative way (22). In this
regard, a similar situation is present in T. maritima, in
which the
-fucosidase gene is part of a cluster of six ORFs
potentially involved in xyloglucan utilization (22); remarkably,
S. solfataricus, strain P2, hydrolyzes xyloglucan from
tamarind seed, which is not
fucosylated.3 The ability of
Ss
-fuc to hydrolyze short fucosylated oligosaccharides is revealed
by the rapid hydrolysis observed using
-L-Fuc-(1-3)-
-L-Fuc-O-pNp substrate. The hypothesis that Ss
-fuc, XylS, and Ss
-gly cooperate in the sequential hydrolytic steps on fucosylated xyloglucans from
plant cell walls (38) is tempting and is currently under investigation,
in the hope of shedding some light on a novel metabolic pathway in
S. solfataricus for the utilization of this hemicellulose as
a carbon source.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to V. Salerno for the total RNA preparations. We thank V. Carratore and L. Camardella for protein sequencing.
![]() |
FOOTNOTES |
---|
* This work was supported by Agenzia Spaziale Italiana Project "Extremophilic Archaea as Model Systems to Study Origin and Evolution of Early Organisms: Molecular Mechanisms of Adaptation to Extreme Physical-Chemical Conditions" Contract I/R/365/02 and by MIUR Project RBAU015B47 "Folding di Proteine: l'Altra Metà del Codice Genetico."The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This paper is dedicated to the memory of Eraldo Antonini, eminent biochemist, prematurely deceased 20 years ago, on March 19th, 1983.
The on-line version of this article (available at
http://www.jbc.org) contains the NMR spectra
of the products obtained.
To whom correspondence should be addressed: Institute of
Protein Biochemistry-CNR, Via P. Castellino 111, 80131, Naples, Italy. Tel.: 39-081-6132271; Fax: 39-081-6132277; E-mail:
moracci@dafne.ibpe.na.cnr.it.
Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M211834200
2 B. Cobucci-Ponzano, M. Rossi, and M. Moracci, manuscript in preparation.
3 B. Cobucci-Ponzano, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GH29, family 29 of
glycosyl hydrolases;
ORF, open reading frames;
Ss-fuc, mutant
S. solfataricus
-fucosidase;
pNp-Fuc, p-nitrophenyl-
-L-fucopyranoside;
pNp-Glc, p-nitrophenyl-
-D-glucoside;
GST, glutathione
S-transferase;
XylS, S. solfataricus
-xylosidase;
Ss
-gly, S. solfataricus
-glycosidase;
PBS, phosphate-buffered saline;
RT, reverse transcriptase.
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