Structural and kinetic insights reveal that the amino acid pair Gln-228/Asn-254 modulates the transfructosylating specificity of Schwanniomyces occidentalis β-fructofuranosidase, an enzyme that produces prebiotics

Article abstract of DOI:10.1074/jbc.M112.355503

Schwanniomyces occidentalis β-fructofuranosidase (Ffase) is a GH32 dimeric enzyme that releases fructose from the nonreducing end of various oligosaccharides and essential storage fructans such as inulin. It also catalyzes the transfer of a fructosyl unit to an acceptor producing 6-kestose and 1-kestose, prebiotics that stimulate the growth of bacteria beneficial for human health. We report here the crystal structure of inactivated Ffase complexed with fructosylnystose and inulin, which shows the intricate net of interactions keeping the substrate tightly bound at the active site. Up to five subsites were observed, the sugar unit located at subsite +3 being recognized by interaction with the β-sandwich domain of the adjacent sub-unit within the dimer. This explains the high activity observed against long substrates, giving the first experimental evidence of the direct role of a GH32 β-sandwich domain in substrate binding. Crucial residues were mutated and their hydrolase/transferase (H/T) activities were fully characterized, showing the involvement of the Gln-228/Asn-254 pair in modulating the H/T ratio and the type β(2-1)/β(2-6) linkage formation. We generated Ffase mutants with new transferase activity; among them, Q228V gives almost specifically 6-kestose, whereas N254T produces a broader spectrum product including also neokestose. A model for the mechanism of the Ffase transfructosylation reaction is proposed. The results contribute to an understanding of the molecular basis regulating specificity among GH-J clan members, which represent an interesting target for rational design of enzymes, showing redesigned activities to produce tailor-made fructooligosaccharides.

Full text of DOI:10.1074/jbc.M112.355503

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 23, pp. 19674–19686, June 1, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Structural and Kinetic Insights Reveal That the Amino Acid
Pair Gln-228/Asn-254 Modulates the Transfructosylating
Specificity of Schwanniomyces occidentalis
□
S
␤-Fructofuranosidase, an Enzyme That Produces Prebiotics*
Received for publication, February 23, 2012, and in revised form, April 13, 2012 Published, JBC Papers in Press, April 16, 2012, DOI 10.1074/jbc.M112.355503
Miguel Álvaro-Benito‡1,2, M. Angela Sainz-Polo§2, David González-Pérez‡, Beatriz González§, Francisco J. Plou¶,
María Fernández-Lobato‡3, and Julia Sanz-Aparicio§4
From the ‡Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular, Universidad Autónoma de
Madrid-Consejo Superior de Investigaciones (UAM-CSIC), Cantoblanco, 28049 Madrid, Spain, the §Departamento de
Cristalografía y Biología Estructural, Instituto de Química-Física “Rocasolano”-CSIC, Serrano 119, 28006 Madrid, Spain,
and the ¶Instituto de Catálisis y Petroleoquímica-CSIC, 28049 Madrid, Spain
Background: Schwanniomyces occidentalis ␤-fructofuranosidase synthesizes 6-kestose and 1-kestose, prebiotics that stim-
ulate beneficial bacteria.
Results: The ␤-sandwich domain is involved in substrate binding, and the Gln-228/Asn-254 pair plays a crucial role in acceptor/
donor substrate binding and product specificity.
Conclusion: First evidence of ␤-sandwich domain functionality. Variants with new specificities, synthesizing neokestose, were
obtained.
Significance: Understanding the molecular mechanism that regulates product specificity is crucial for designing improved new
enzymes.
Schwanniomyces occidentalis ␤-fructofuranosidase (Ffase) is N254T produces a broader spectrum product including also
a GH32 dimeric enzyme that releases fructose from the nonre- neokestose. A model for the mechanism of the Ffase transfruc-
ducing end of various oligosaccharides and essential storage tosylation reaction is proposed. The results contribute to an
fructans such as inulin. It also catalyzes the transfer of a fructo- understanding of the molecular basis regulating specificity
syl unit to an acceptor producing 6-kestose and 1-kestose, pre- among GH-J clan members, which represent an interesting tar-
biotics that stimulate the growth of bacteria beneficial for get for rational design of enzymes, showing redesigned activities
human health. We report here the crystal structure of inacti- to produce tailor-made fructooligosaccharides.
vated Ffase complexed with fructosylnystose and inulin, which
shows the intricate net of interactions keeping the substrate
Fructans and fructooligosaccharides (FOS)5 are ␤-D-fructose
(Fru)-based carbohydrates, often linked to a sucrose skeleton,
that are synthesized by 15–20% of the flowering plants and also
by a number of bacteria and fungi. These compounds are
related to carbohydrate storage and the processes of drought
and freezing tolerance in plants (1). In addition, FOS are con-
sidered prebiotics because they selectively stimulate the
growth/activity of Lactobacilli and Bifidobacteria from the ani-
mal digestive tract (1, 2) and exert a beneficial effect on human
health, thus contributing to the prevention of cardiovascular
diseases, colon cancer, and osteoporosis (3). Different types of
FOS series can be distinguished depending on the linkage type
between the monosaccharide residues: 1F-FOS, containing
␤-(231)-linked fructose units (with an inulin-type structure,
e.g. 1-kestose or nystose); 6F-FOS, containing ␤-(236)-linked
fructose units (with a levan-type structure, e.g. 6-kestose); and
6G-FOS, where the ␤-(236)-link connects fructose and the
glucosyl moiety of sucrose (neo-FOS, e.g. neokestose or neonys-
tightly bound at the active site. Up to five subsites were
observed, the sugar unit located at subsite ؉3 being recognized
by interaction with the ␤-sandwich domain of the adjacent sub-
unit within the dimer. This explains the high activity observed
against long substrates, giving the first experimental evidence of
the direct role of a GH32 ␤-sandwich domain in substrate bind-
ing. Crucial residues were mutated and their hydrolase/trans-
ferase (H/T) activities were fully characterized, showing the
involvement of the Gln-228/Asn-254 pair in modulating the
H/T ratio and the type ␤(2–1)/␤(2–6) linkage formation. We
generated Ffase mutants with new transferase activity; among
them, Q228V gives almost specifically 6-kestose, whereas
* This work was supported by Grants BIO2010-20508-C04-01, -03, and -04, as
wellasBIO2007-67708-C04-01, -03, and-04, from the Ministryof Education
and Science, Spain.
□S
This article contains supplemental Table S1.
The atomic coordinates and structure factors (codes 3U14 and 3U75) have been
deposited in the Protein Data Bank, Research Collaboratory for Structural
Bioinformatics, Rutgers University, NewBrunswick, NJ(http://www.rcsb.org/).
1 Supported by the Spanish Comunidad de Madrid program.
2 Both authors contributed equally to this work.
5 The abbreviations used are: FOS, fructooligosaccharide(s); FEH, fructan exo-
hydrolase; Ffase, ␤-fructofuranosidase from Schwanniomyces occidentalis;
FT, fructosyltransferase; GH, glycosylhydrolase; H/T, hydrolase/transferase
activityratio;PDB, ProteinDataBank;CSD, CambridgeStructuralDatabase;
MPD, 2-methyl-2,4-pentanediol.
3 To whom correspondence may be addressed. Tel.: 34-91-196-4492; Fax:
34-91-196-4420; E-mail: mfernandez@cbm.uam.es.
4 To whom correspondence may be addressed. Tel.: 34-91-561-9400; Fax:
34-91-564-2431; E-mail: xjulia@iqfr.csic.es.
19674 JOURNAL OF BIOLOGICAL CHEMISTRY
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Sw. occidentalis ␤-Fructofuranosidase Specificity
FIGURE 1. General reaction catalyzed by GH-J clan enzymes through a double displacement mechanism that involves a covalent intermediate. The
␤-(231)-linked fructan represented (the donor substrate) would be hydrolyzed in the second step of the reaction when water is the acceptor substrate of the
fructosyl (r ϭ H). In the case of an oligosaccharide acting as acceptor, a transfructosylating product is obtained. The inulin-type scaffold is: sucrose (n ϭ 0),
1-kestose (n ϭ 1), nystose (n ϭ 2), fructosylnystose (n ϭ 3), inulin (n Ϸ 30–50).
tose). Although the molecules included in the three series show phile transform a plant cell wall invertase into a fructosyltrans-
prebiotic effect (4, 5), ␤-(236)-linked FOS may have enhanced ferase (19, 20), whereas a single substitution in a fructan 6-exo-
prebiotic properties and chemical stability as compared with hydrolase (F233D) turns it into a fructan 1-exohydrolase (21).
1F-FOS (4). Enzymatic synthesis of these oligosaccharides has Moreover, only three amino acids (Asn-340, Trp-343, and Ser-
been reported using different enzymes from microorganisms. 415) appear to be responsible for donor substrate specificity
Thus, the ␤-fructofuranosidases from Saccharomyces cerevi- in 6-glucosyl-1-fructosyl-fructosyltransferase from Lolium
siae (6), Schwanniomyces occidentalis (7, 8), and Rhodotorula perenne (22). Within this framework, the first three-dimen-
dairenensis (9) produce mainly 6-kestose, whereas the enzymes sional structure of a GH32 fructosyltransferase (FT) in complex
from Xanthophyllomyces dendrorhous synthesize neokestose with nystose (a medium size substrate) has already been
(10, 11).
reported (16), and in agreement with previous works (21), it was
On the basis of their amino acid sequences, fructans process- postulated that residues in binding subsites ϩ1 and ϩ2
ing enzymes are classified into the glycoside-hydrolase (GH) (nomenclature according to Davies et al. (23)) seem to be
GH-J clan, in which two families are included: GH32 (inverta- responsible for the specificity of the products formed in the
ses, ␤-fructofuranosidases, inulinases, and fructosyltrans- transfructosylation reaction.
ferases) and GH68 (inulosucrases and levansucrases) (12). GH-J
The ␤-fructofuranosidase (Ffase) from the yeast Sw. occiden-
clan enzymes share a five-blade ␤-propeller N-terminal talis hydrolyzes sucrose, 1-kestose, nystose, or inulin and effi-
domain, in which ␤-sheets are arranged around a central ciently produces the trisaccharides 6-kestose and 1-kestose (in
pocket that accommodates the active site (catalytic domain). the ratio of 3:1); both are prebiotic FOS (7). The Ffase three-
Three key acidic residues located in the active site and sur- dimensional structure has been solved as a homodimer, each
rounded by conserved sequences in the GH32 family, NDPNG modular subunit arranged like other GH32 enzymes including
(Asp (D) acting as nucleophile), RDP (Asp (D) acting as a stabi- a N-terminal ␤-sandwich domain attached to the catalytic
lizer of the transient state) and EC (Glu (E) acting as the acid ␤-propeller. Interestingly, the active site cleft is shaped by both
base catalyst), are implicated in substrate binding and hydroly- subunits within the dimer, which seem to be involved in sub-
sis (13). These enzymes display either hydrolysis or transfruc- strate binding (15). The Asp-50 (NDPNG) and Glu-230 (EC)
tosylation reactions (Fig. 1) depending on whether water or an located at the center of the ␤-propeller are the catalytic residues
oligosaccharide (the acceptor substrate) molecule accepts the implicated in substrate binding and hydrolysis, whereas Arg-
fructose released by fructan hydrolysis (the donor substrate). 178 and Asp-179 form the RDP motif. The structural analysis of
The main differences between proteins included in GH32 and Ffase active site, in combination with a mutational and kinetic
GH68 are the presence of an additional ␤-sandwich C-terminal analysis, has recently suggested the existence of a novel donor
domain in the former and the general transfructosylating char- substrate binding site located in the EC motif environment
acter of the latter. The number of studies giving insight into the (including residues such as Ser-196, Gln-176, Gln-228, Pro-
structural determinants involved in GH-J clan specificity has 232, and Asn-254) that presumably is responsible for the trans-
grown significantly in recent years (14–18). These studies show ferase specificity of the enzyme (8). In this article we report the
that the loops and turns connecting the different elements of crystal structure of two different Ffase inactivated mutants
the ␤-propeller domain, in the environs of the active site, seem complexed with medium and long chain substrates, giving the
to define the specificity unique to each enzyme. Thus, only a few first experimental evidence of the direct role of a GH32 supple-
substitutions in the vicinity of the residue acting as a nucleo- mentary ␤-sandwich domain in substrate binding. Moreover,
JUNE 1, 2012•VOLUME 287•NUMBER 23
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Sw. occidentalis ␤-Fructofuranosidase Specificity
potential oligosaccharide-binding residues were mutated and around 30 g of cells (expressing each inactive variant) were
their hydrolase/transferase (H/T) activities characterized. Our
results directly and unambiguously associate the specificity of
the Ffase transferase activity with residues located in the envi-
ronment of Glu that act as the acid/base catalyst in the GH32
catalytic mechanism.
lysed by the addition of YeastBuster reagent (Novagen) includ-
ing the protease inhibitor mixture for fungal and yeast extracts
(Sigma) as indicated by the manufacturers. The cellular debris
was removed by centrifugation, and the supernatant was fitted
to 40% glycerol, 0.5% Tween 20, 300 mM NaCl, and 20 mM
sodium phosphate, pH 8.0. The samples were then incubated
for 2 h at 4 °C with nickel-nitrilotriacetic acid-agarose (Qiagen)
(1 ml of resin gϪ1 cells) using an orbital shaker and applied to a
suitable column support as recommended by the manufac-
turer. No retained proteins were eluted using 8 mM imidazole.
The protein of interest eluted in 50 mM sodium acetate, pH 4.5.
All fractions were analyzed by Western blot as described below.
Fractions containing Ffase protein were dialyzed in 20 mM HCl-
Tris, pH 7.0, and concentrated by using the Microcon YM-10
(Amicon) system.
EXPERIMENTAL PROCEDURES
Materials, Organisms, Plasmids, and Mutagenesis—Nystose
(␣-D-glucopyranosyl-[(132)-␤-D-fructofuranosyl]2-(132)-
␤-D-fructofuranose), fructosylnystose (␣-D-glucopyranosyl-
[(132)-␤-D-fructofuranosyl]3-(132)-␤-D-fructofuranose), and
inulin from chicory (␣-D-glucopyranosyl-[(132)-␤-D-fructo-
furanosyl]n-(132)-␤-D-fructofuranose); n-36) were obtained
from TCI-Europe, Megazyme, and Sigma-Aldrich, respec-
tively. S. cerevisiae EUROSCARF Y02321 (BY4741; Mat a;
his3⌬1; leu2⌬0; met15⌬0; ura3⌬0; YIL162w(SUC2)::kanMX4)
was used as the expression host, and it was transformed by the
standard lithium acetate method. Escherichia coli DH5␣
Western blot analysis was carried out using standard proce-
dures and probed with anti-His6 antibody (Bio-Rad) as indi-
cated by the manufacturer and/or the anti-S. cerevisiae invert-
ase antibody, which was obtained and used as indicated
previously (8).
Ϫ
(␭ ␾80dlacZ⌬M15 ⌬(lacZYA-argF)U169 recA1 endA1 hsdR17-
Ϫ
Ϫ
(rK mK ) supE44 thi-1 gyrA relA1) was used for DNA manipula-
tion and amplification by standard techniques.
Enzyme and Kinetic Analysis—Ffase hydrolytic activity was
quantified by the dinitrosalicylic acid method adapted to a
96-well microplate as described previously (7). Enzymatic
assays were performed in 100 mM sodium acetate buffer, pH
5.0, at 50 °C. One unit of activity was defined as that catalyzing
the formation of 1 ␮mol of reducing sugar/min. For kinetic
analysis, the initial velocity was measured in triplicate using
0.3–5.3 ␮g mlϪ1 of enzyme, sucrose, or nystose (2.5–100 mM)
and inulin (1–10 mM). The reaction time was 20 min. Plotting
and analysis of the curves were carried out using SigmaPlot
software (version 7.101). Kinetic parameters were calculated by
fitting the initial rate values to the Michaelis-Menten equation.
To study the effect of sucrose concentration on transfructo-
sylation activity, the concentration of sucrose was varied
between 250 and 1000 mM at a standard reaction time of 3 h and
47.5 °C. For the analysis of the selectivity of transfructosylation
products (6-kestose, 1-kestose, and neokestose), 1 M sucrose
and a 24-h reaction time were used. Reactions were stopped (5
min at 100 °C), and the carbohydrate concentration was deter-
mined by high-performance liquid chromatography (HPLC) as
indicated previously (7).
The Ffase gene (CQ890277) from Sw. occidentalis ATCC
26077 used in this work was included in the construct Ffase-
pYES and contained the only encoding CTG triplet (positions
ϩ586 to ϩ588) mutated to TCA (L196S), which encodes serine
in the standard code to obtain a heterologously expressed pro-
tein with the wild-type amino acid sequence (8). This construct
was used as template to obtain all of the enzymatic mutants
generated and analyzed in this work. Site-directed mutagenesis
was carried out using specific primers (supplemental Table S1)
and the method described previously (15). The full-length
Ffase, including the substitution D50A or E230A fused to a
C-terminal His6 tag, was obtained using the Ffase-pYES D50A
or E230A construct as template and the oligonucleotides SwF-
BamHI (5Ј-TAGGATCCAACATGGTACAAGTTTTAAGT-
GTATTAG-3Ј) and SwRHXbaI (5Ј-CCCTCTAGACTTATT-
TAGTTCCCTTATGATAAC-3Ј). Restriction sites for BamHI
and XbaI (shown in bold) were included in these primers to
clone the PCR products into the pYES2/CT (Invitrogen) vector,
thereby generating the Ffase-pYESCT D50A and E230A con-
structs. DNA sequencing (SIDI, Universidad Autónoma de
Madrid, Spain) was used to verify that only the desired muta-
tions were present in all of the constructs obtained.
Crystallization and Data Collection—As deglycosylation of
wild-type Ffase increased the diffraction resolution limit from
2.9 to 1.9 Å (15), the recombinant proteins expressed by the
Ffase-pYESCT D50A and E230A constructs were submitted to
treatment with endoglycosidase H (New England Biolabs),
which yielded a unique protein band below 66 kDa, as observed
from the SDS-PAGE analysis performed for both mutant pro-
teins (data not shown). The samples were concentrated, and
screening of new crystallization conditions was performed with
the PACT suite from Qiagen. Plate-shaped crystals appeared in
solutions containing either PEG 3350 or PEG 6000 at pH 6 or 8.
Further optimization was performed at room temperature by
the vapor diffusion method by mixing 1 ␮l of protein solution
with an equal volume of different reservoir solutions and using
Protein Purification and Detection—␤-Fructofuranosidase
mutants from Sw. occidentalis expressed in S. cerevisiae were
purified as described elsewhere (8). Basically, yeasts were grown
in YPGal medium (1 liter), and the culture filtrates were con-
centrated and applied to a DEAE-Sephacel chromatography
column and eluted using a 0–0.5 M NaCl gradient. Samples
containing fructouranosidase activity eluted in 0.1 M NaCl and
were analyzed by Coomassie-stained SDS-PAGE (8%). Only
those containing apparently pure proteins were pooled, dia-
lyzed, concentrated, and stored at Ϫ70 °C. Protein concentra-
tion was estimated photometrically at 280 nm (NanoDrop
spectrophotometer ND-1000).
His-tagged proteins (Ffase including D50A or E230A substi-
tution) were purified from yeast cellular fractions. Basically, the sitting-drop procedure.
19676 JOURNAL OF BIOLOGICAL CHEMISTRY
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Sw. occidentalis ␤-Fructofuranosidase Specificity
The best crystals of the E230A mutant concentrated to 10.5 His6 tag was actively expressed in S. cerevisiae, but initial
mg mlϪ1 grew from 16% PEG 6000, 3% MPD, 0.2 M MgCl2, and attempts to obtain good quality crystals from this recombinant
0.1 M MES, pH 6, and reached the maximum size in 10–15 days. enzyme were unsuccessful (34). Therefore, the three-dimen-
To obtain the E230A-fructosylnystose complex, crystals were sional structure of the enzyme was solved from extracellular
transferred to a soaking solution containing 19% PEG 6000, 3% enzyme purified directly from Sw. occidentalis. To determine
MPD, 0.1 M MES, pH 6, and 100 mM fructosylnystose for 5 min, the crystal structure of the substrate complexes, the two cata-
and subsequently to a fresh drop supplemented with 20% glyc- lytic residues, Asp-50 and Glu-230, were changed to Ala, a
erol prior to cooling to 100 K in a nitrogen stream. A full data set change that previously had been demonstrated to yield variants
was collected to 2.68 Å at the ESRF ID23-2 beamline.
with undetectable activity against both short (sucrose) as well as
Plate-like crystals of the D50A mutant concentrated to 11.2 medium and long size (nystose and inulin) substrates (15).
mg mlϪ1 grew from 15% PEG 6000, 3% MPD, 0.2 M MgCl2, and These mutated variants of Ffase were submitted to deglycosy-
0.1 M HCl-Tris, pH 8. The D50A-inulin complex was obtained lating digestion with Endo H, which proved to be crucial in
by soaking a crystal in 17% PEG 6000, 3% MPD, 0.1 M MES, pH getting good crystals. The best crystals from both mutants were
6, and 2.65% inulin for 4 h before being cryoprotected in a drop used for soaking experiments, and finally the complexes D50A-
supplemented with 20% glycerol to be flash-frozen to 100 K. A inulin and E230A-fructosylnystose were solved. In accordance
full data set was collected at the ESRF ID14-1 beamline.
with the deglycosylated wild type, the recombinant Ffase
Analysis of the diffraction patterns was performed using D50A-inulin complex belongs to the P21 space group, with two
MOSFLM (24) to yield integrated intensities, and data were molecules in the asymmetric unit, whereas the E230A-fructo-
merged with the CCP4 suite (25). A summary of data collection sylnystose complex loses symmetry, with the crystal showing
and data reduction statistics is shown in Table 1.
the P1 space group and four molecules in the asymmetric unit.
Structure Solution and Refinement—The structure of the The structure of both complexes was solved by molecular
complexes was solved using the molecular replacement replacement with the coordinates of native Ffase and refined to
method. Initial phases were obtained with the MOLREP pro- the final values given in Table 1. The initial difference maps
gram (26) using the atomic coordinates of deglycosylated wild- revealed unequivocally the presence of the substrates in all of
type Ffase (PDB code: 3KF3) as the search model. A clear solu- the active sites that were refined, showing apparently full occu-
tion was obtained in both mutants, which was followed by pancies. The final electron density of both substrates, together
crystallographic refinement using Refmac (27) in the CCP4 with the folding of the Ffase dimer, is shown in Fig. 2. Several
suite (25) with flat bulk-solvent correction, including low reso- N-acetylglucosamine units, also visible, were attached to Asn-
lution data to 50 Å, and using maximum likelihood target fea- 72, Asn-126, Asn-219, Asn-334, and Asn-394.
tures. A subset of 5% randomly selected structure-factor ampli-
Overall, the crystal structure of both mutants (Fig. 2) is essen-
tudes was excluded from automated refinement to compute a tially identical, as revealed by the root-mean-square deviation
free R-factor. Several rounds of refinement applying non-crys- of 0.321 Å between their equivalent C␣ atom coordinates. They
tallographic symmetry restraints between the molecules in the are also mostly similar with respect to the wild type, the D50A-
asymmetric unit were alternated with rebuilding using the pro- inulin, and E230A-fructosylnystose complexes, showing root-
gram O (28) after which the Fo Ϫ Fc maps showed unequivo- mean-square deviation values of 0.303 and 0.279 Å, respec-
cally the positions of the substrates, well ordered at the active tively, as superimposed on the same C␣ atoms of the
site. The inulin chain coordinates were retrieved from the crys- deglycosylated wild-type structure (PDB code: 3KF3). On the
tal structure determined from electron diffraction data (29) and other hand, there were no significant alterations in the active
deposited in the Cambridge Structural DatabaseCSD; Refcode site upon complexion, apart from a change in the conformation
VIPRAN), whereas the fructosylnystose was modeled manually of the catalytic Glu-230 side chain and some minor differences
on the basis of the nystose coordinates retrieved from the crys- in the orientation of the aromatic Trp-47, Tyr-293, Trp-310,
tal structure (30) deposited in the CSD (Refcode PEKHES). and Trp-314 side chains, all of them located at the active site.
Both substrates were fitted manually into the electron density
Ffase Dimer Induces a Fixed Conformation of the Substrate—As
and further refined. At the last stage, water molecules and sev- shown previously, each active site of the Ffase dimer is made up
eral N-acetylglucosamine were included, and further model from both subunits that contour a cavity 25 Å deep, accessible
completion was performed with the program Coot (31) com- to the solvent by a slot 18 Å wide. Fig. 3A displays a close-up
bined with more rounds of positional and individual restrained view of this cavity with the bound substrates observed in the
B-factor refinement. This led to a final R-factor of 0.19 (Rfree ϭ complexes. We observed that the non-reducing end of the sub-
0.23) for the D50A-inulin complex data set up to 2.24 Å reso- strates was hidden deeply inside the catalytic pocket, as
lution and 0.23 (Rfree ϭ 0.31) for the E230A-fructosylnystose expected, but the remaining sugar units were also well inserted
data set up to 2.68 Å. Final refinement parameters for both into the cavity and well adjusted to both subunits within the
complexes are reported in Table 1. The stereochemistry of the dimer. A subsequent fructose ring of inulin should be dangling
model was checked by using ProCheck (32), and the figures out into the solvent with no atomic interactions to the protein
were generated with PyMOL (33).
and, consequently, not visible in the electron density maps.
Remarkably, both oligosaccharides adopt a very similar confor-
mation along the fructose-fructose bonds, as illustrated in Fig.
RESULTS
Ffase Inactive Mutants Trap the Substrates—Prior to this 3B, the only differences being observed in the glucose moiety at
work, the Ffase from Sw. occidentalis (also SoInv) fused to a the reducing end of fructosylnystose, which in any case is
JUNE 1, 2012•VOLUME 287•NUMBER 23
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Sw. occidentalis ␤-Fructofuranosidase Specificity
TABLE 1
X-ray data collection and structure refinements statics
Crystal
D50A-Inu
E230A-Fnys
Data collection
Unit cell dimensions (Å)
Space group
67.1, 108.6, 96.1 (␤ ϭ 91.4°)
65.21, 92.66, 117.75 89.91, 82.96, 85.82
P21
P1
Molecules in asymmetric unit
Solvent content (%)
2
4
42
42
Limiting resolution (Å) (outer shell)
Unique reflections
2.24 (2.36–2.24)
65,603
2.68 (2.82–2.68)
74,146
a
Rrim
0.14 (0.51)
0.07 (0.24)
98.4 (98.2)
5.2 (5.0)
5.3 (1.5)
0.22 (0.44)
0.11 (0.44)
96.4 (88.4)
4.3 (2.0)
8.3 (2.9)
b
Rpim
Completeness (%)
Mean multiplicity
Mean I/␴I
Final refinement parameters
Protein atoms (non-H)
Solvent molecules
Ligand molecules
Glycosylation molecules
R-factorc
8,100
16,200
623
279
2 Fru6
4 Fructosylnystose
8 N-acetylglucosamine
20 N-acetylglucosamine
0.19
0.23
Rfree
0.23
0.31
r.m.s.d bonds (Å)/angles (°)
Averaged B-factors (Å2)
Main chain
0.012/1.8
0.014/1.8
15.6
16.5
31.2
23.2
32.8
33.1
30.0
45.6
Side chain
Ligand
Solvent
Ramachandran plot
Favored (%)
98.8
1.2
96.7
3.3
Outliers (%)
Protein Data Bank code
3U14
3U75
a Rrim ϭ ⌺[N/(N Ϫ 1)]1/2⌺͉I(h)I Ϫ ͗I(h)͉͘]/⌺⌺͗I(h)͘.
b Rpim ϭ ⌺[1/(N Ϫ 1)]1/2⌺͉I(h)i Ϫ ͗I(h)͉͘/⌺⌺͗I(h)͘.
c R-factor ϭ ⌺(͉Fobs Ϫ Fcalc͉)/⌺͉Fobs͉ (Rfree is equivalent to R-factor for a randomly selected 5% subset of reflections not used in structure refinement).
d r.m.s., root mean square.
located in an equivalent position to the fifth fructose unit of
inulin. This means that the substrates are accommodated into
the Ffase cavity in a very precise conformation that is imposed
by a highly specific catalytic active site, which is able to recog-
nize oligosaccharide units distal from the scissile bond.
On the other hand, the structure of bound inulin is very
similar to the helical conformation observed in its free crys-
talline state (29), with only small differences observed at the
last visible fructose unit (Fig. 3C). On the contrary, the con-
formation of fructosylnystose differs considerably from that
found in crystallized free nystose (30) and nystose bound to
Aspergillus japonicus FT (16) as shown in Fig. 3D. Therefore,
it is apparent that the molecular surface of the dimer has
evolved to accommodate the polymeric chain of inulin. The
fact that dimerization of this enzyme is involved in the deg-
radation of inulin and other long chain substrates has been
proposed previously by us (15). Furthermore, and consider-
ing the Ffase catalytic efficiency on sucrose (S) and inulin (I)
that gives an S/I ratio of 12, its catalytic behavior (Table 2)
seems to be more typical of that of an inulinase (S/I ratio Ͻ
10) as compared with that expected from an invertase (S/I
ratio Ͼ 1600) (35). Inulinases are generally monomeric
enzymes with a molecular mass ranging from 50 to 70 kDa
(36), although a few multimeric proteins such as that from
the yeast Kluyveromyces fragilis (37) or the fungi Aspergillus
fumigatus (38) have also been described. Unfortunately, no
functional role for the oligomerization of these enzymes has
been assigned, making the Ffase the unique enzyme
described to date for which functionality and oligomeriza-
tion have been correlated.
FIGURE 2. Structure of the Ffase complexes. A, overall structure of Ffase
dimer is represented in ribbon diagram, with two inulin chains found in the
D50A crystals represented as spheres and the glycosylation units represented
as sticks. Each subunit (cyan and orange) is bimodular and consists of a cata-
lytic ␤-propeller domain (dark) and a C-terminal ␤-sandwich domain (light).
BandC, close-upviewofD50A-inulin(B)andE230A-fructosylnystose(C)com-
plex binding sites. The colored portion of the 2Fo Ϫ Fc electron density map at
2.2 Å covering the inulin (B) and 2.7 Å covering the oligosaccharide (C) is
contoured at 1.0 ␴ level.
19678 JOURNAL OF BIOLOGICAL CHEMISTRY
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Sw. occidentalis ␤-Fructofuranosidase Specificity
Ffase Accommodates up to Five Sugar Units in Its Active Site— site reveals that the enzyme presents five binding subsites, i.e. it
A detail of the inulin and fructosylnystose recognition mode by
FfaseisdisplayedinFig. 4. Inspectionofthecomplexesattheactive
delineates subsites Ϫ1 to ϩ4, following the nomenclature used for
sugar-binding substrates (23) that defines cleavage as occurring
between subsites Ϫ1 and ϩ1. The subsites are made up from res-
idues in all five blades of one subunit and also from the ␤-sandwich
domain of the adjacent subunit. These residues establish a net of
hydrogen bonding, some of them through well ordered water mol-
ecules, which keeps the substrates in a very fixed conformation. In
addition, even when none of the stacking interaction typically
found in protein-sugar interactions is found, several aromatic res-
idues (Trp-47, Trp-76, Phe-110, and Trp-314) define a hydropho-
bic patch at one wall of the active site cavity (see Fig. 4) that directs
the ␤-1,2 glycosidic bonds of the oligosaccharide.
The fructose unit bound at subsite Ϫ1 is tightly fixed by the
hydrogen bonds of all of its hydroxyl groups to Asn-49, Gln-68,
Trp-76, Arg-178, and Asp-179 side chains and to both the main
and side chains of Ser-111. The only difference found between
the two complexes is the fructosylnystose O1 that is hydrogen-
bonded to Trp-314 and the nucleophile Asp-50 side chains,
whereas in the D50A mutant a water molecule occupies a posi-
tion close to the removed carboxylate and links the O1 to the
Ala-50 main chain. Apart from this feature due to the removal
of the Asp-50 side chain, the interaction pattern at subsite Ϫ1 is
highly conserved along the GH32 complexes.
At subsite ϩ1, the fructose O3 atom makes direct hydrogen
bonds to the side chains of Glu-230, Gln-228, and Arg-178, with
the last also interacting with O4. Furthermore, there are two
highly ordered water molecules that link both hydroxyls to
these residues, and also to other residues such as Gln-176 and
Asn-254, in a net of polar interactions that keeps the fructose
ring in a very fixed orientation. Also, the glycoside links by
hydrogen bond to its fructose O2. In turn, the fructose unit
located at subsite ϩ2 makes hydrogen bonds at O3 to the Asn-
254 side chain and at O4 to Asn-227, the latter through a water
molecule. Moreover, there is an intramolecular link between
the O6 of the fructose units, located at ϩ2 and ϩ3, through a
water molecule in the case of the complex with inulin (Fig. 4).
Subsite ϩ3 is defined mainly by subunit B through hydrogen
bonds of the Ser-434 main chain and the side chain of Gln-435
to fructose O6 and O4 atoms, respectively. Additional links are
made with subunit A through the main chain of Trp-314 to O4
and Asp-318 and Arg-451 side chains to O3 and O4, respec-
tively. In contrast, subsite ϩ4 is lined up only by residues from
FIGURE 3. The conformation of the substrates at the Ffase active site. A,
close-up view of the Ffase active site showing the catalytic domain of one
subunit colored in cyan and the C-terminal ␤-sandwich domain of the adja-
cent subunit in beige. The six units of inulin (left) and fructosylnystose (right)
molecules found in the Ffase inactivated mutants are represented as spheres.
B, inulin (brown) and fructosylnystose (lime green) moieties, in stick represen-
tation, aresuperimposedintwoperpendicularviewstohighlightthefactthat
both substrates adopt the same conformation at the Ffase active site. C, inulin
found at the D50A crystal (brown) is compared with inulin found in its free
crystalline state (white). D, fructosylnystose found in the Glu-230 crystal (lime
green) is superimposed on nystose found in the A. japonicus fructosyltrans-
ferase complex (green) and crystallized free nystose (white), showing that
they adopt very different conformations.
TABLE 2
Kinetic analysis of Ffase mutants
The kcat values were calculated assuming a protein molecular mass of 180 kDa. The Ϯ sign refers to standard errors based on curve fitting using SigmaPlot.
kcat
Km
kcat/K
m
Ffase
mutant
Sucrose
Nystose
Inulin
Sucrose
Nystose
mM
Inulin
Sucrose
Nystose
Inulin
Ϫ1
Ϫ1
s
s
Ϫ1mM
WT
105 Ϯ 14
144 Ϯ 28
32 Ϯ 3
82 Ϯ 11
28 Ϯ 5
29 Ϯ 4
64 Ϯ 11
38 Ϯ 6
80 Ϯ 9
10 Ϯ 1
31 Ϯ 6
16 Ϯ 1
99 Ϯ 6
208 Ϯ 7
50 Ϯ 7
91 Ϯ 2
1.8 Ϯ 0.2
1.8 Ϯ 1.9
30 Ϯ 3.3
25 Ϯ 3
61 Ϯ 3
40 Ϯ 8
82 Ϯ 3
72 Ϯ 2
19.9 Ϯ 3
1220 Ϯ 20
53.8 Ϯ 6
18.5 Ϯ 7
3.4 Ϯ 0.2
5.0 Ϯ 4.5
91 Ϯ 8.1
409 Ϯ 40
11.3 Ϯ 0.9
2.1 Ϯ 0.2
4.4 Ϯ 0.4
1.3 Ϯ 0.3
6.4 Ϯ 0.9
6.0 Ϯ 0.8
6.7 Ϯ 0.5
8.2 Ϯ 1
2.1 Ϯ 0.4
30 Ϯ 1.4
2.9 Ϯ 0.2
2.5 Ϯ 0.3
9.4 Ϯ 0.9
11.3 Ϯ 2
1.0 Ϯ 0.2
1.2 Ϯ 0.4
9.2 Ϯ 2
14.7 Ϯ 2.4
814 Ϯ 8
16.4
24
47.1
6.9
1.3
Q176S
Q176N
Q176E
Q228V
Q228T
Q228N
Q228E
N254A
N254T
N254D
N254E
1.5
102 Ϯ 9
4.8
10
17.2
36.4
0.2
0.5
20.5 Ϯ 2.7
52.5 Ϯ 7.9
57.1 Ϯ 6
0.9
10.7 Ϯ 2
18.0 Ϯ 8.4
16.1 Ϯ 2.7
3.3 Ϯ 0.5
8.0 Ϯ 0.9
5.0 Ϯ 5.5
13.0 Ϯ 2.4
3.5 Ϯ 0.4
2.6
1.6
4
0.06
0.087
3.8
0.2
23.9 Ϯ 2.8
245 Ϯ 32
8.3 Ϯ 0.8
12.7 Ϯ 1.1
10.2 Ϯ 1.2
8.8 Ϯ 1.3
30
11.5
10
20.8
6.6
1.7
1.3
9 Ϯ 0.3
2
4.4
0.2
4.8 Ϯ 0.7
19.5 Ϯ 2
2.4
4.6
17.1
3.7
0.4
0.1
JUNE 1, 2012•VOLUME 287•NUMBER 23
JOURNAL OF BIOLOGICAL CHEMISTRY 19679

Sw. occidentalis ␤-Fructofuranosidase Specificity
FIGURE 4. Binding mode of the substrates. A, stereo view of the D50A active site with important protein residues engaged in inulin recognition. The major
interactions are indicated by a dotted line. Inulin is colored in brown and residues from the adjacent subunit B in beige. The catalytic triad is represented by
thicker sticks. Water molecules are shown as red spheres. B, stereo view of the Glu-230 active site showing the fructosylnystose in lime green. Direct polar links
between the substrate and the enzyme are shown as dotted lines.
subunit A with the main chain of Trp-76, both through its N 6-kestose (41). With the exception of the A. japonicus FT, the
and O atoms, and with the Asp-318 side chain making hydro- complexes analyzed show that the sugar unit bound at subsite
gen bonds to O3 and O4 of the corresponding fructose unit of ϩ2 is loosely bound, mostly through a few polar interactions
inulin and to the glucose O2 in the fructosylnystose complex. mediated by water molecules, and that additional binding sites
Interestingly, the glucose O2 atom in this complex is located in for longer substrates have been clearly discarded. On the con-
a position equivalent to a well ordered water molecule found in trary, and as it happens with Ffase, the FT from A. japonicus
the complex with inulin that has many interactions with this displays a deep active site able to recognize sugar units from the
substrate. Thus, even when O2 is the only glucose hydroxyl Ϫ1 to ϩ3 subsites (16). However, an inspection of the active site
making polar interactions with residues at the active site, its of both enzymes reveals that only subsite Ϫ1 is conserved,
interaction keeps the glucose tightly bound to the enzyme. In whereas significant differences are found from subsite ϩ1
turn, the corresponding fructose unit from inulin is fixed by toward the reducing end of the substrate.
further additional links to Arg-451 through its O6 atom. Finally,
As seen in Fig. 5, A and B, among all of the aromatic residues
the last visible fructose unit of inulin does not make any direct that shape the hydrophobic wall of the cavity in Ffase, i.e. Trp-
interactions at the active site, which precludes the definition of 47, Trp-76, Phe-110, and Trp-314, only Phe-118 is present in
a ϩ5 subsite and explains why no further inulin chain is evident A. japonicus FT. Furthermore, the terminal fructose unit bound
from the electron density maps.
at subsite ϩ3 in the A. japonicus FT-nystose complex is located
The Catalytic Center of Ffase Differs from Other GH32 exactly in the position corresponding to the side chain of
Members—To date, four GH32 enzymes have been crystallized Trp-76 in Ffase. In fact this part of the A. japonicus FT cavity is
incomplexwithsubstrateslongerthansucrose,a␤-fructofuran- wider because of its lack of loop 70–80 (Ffase numbering),
osidase from Thermotoga maritima with raffinose (39), a plant which nevertheless is conserved in all the GH32 solved up to
fructan exohydrolase (FEH) from Cichorium intybus with present from plants, fungi, and bacteria. On the contrary, the
1-kestose (40), a FT from A. japonicus with raffinose, 1-kestose, cavity at the opposite wall is wider in Ffase than in A. japonicus
and nystose (16), and recently, a FT (6-SST/6-SFT) from Pach- FT, where two loops linking ␤-strands B and C present two long
ysandra terminalis in complex with its acceptor substrate, insertions, residues 140–153 and 320–331, at blades II and IV,
19680 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 23•JUNE 1, 2012

Sw. occidentalis ␤-Fructofuranosidase Specificity
responsible for the transferase activity in A. japonicus FT are
found in the Ffase active site. Moreover, it is worth noting that
the subsites described for A. japonicus FT also diverge signifi-
cantly from the sucrose-binding box motif (WMNDPN),
including the nucleophile Asp, which had been identified as the
binding site for the donor sucrose in plant enzymes and is not
conserved in GH32 FTs.
Finally, in contrast to the deep well found in A. japonicus FT,
which probably precludes the approach of water molecules to
the catalytic pocket favoring the transferase versus the hydro-
lytic activity, Ffase shows a rather wide cleft consistent with its
hydrolytic activity. Furthermore, the polar side chains of Gln-
176, Gln-228, and Asn-254 protrude noticeably at the opposite
face of the hydrophobic wall within this cleft (see Fig. 5A),
delineating a hydrophilic patch that might well be involved in
binding the acceptor sucrose for transferring to fructose, as we
have proposed previously (8). It is worth noting that the Ffase
residues Gln-228/Asn-254 are structurally equivalent to the
Asp-239/Lys-242 pair of Arabidopsis thaliana invertase and
also to the Glu/Arg pair of levansucrases and inolusucrases (Fig.
5C) (17). This is a relevant issue taking into account that the
presence of an acidic residue in this couple seems necessary for
both binding and hydrolysis of sucrose in many clan GH-J
enzymes (14). Therefore, the corresponding Gln/Asn Ffase pair
might play a unique crucial role in its enzymatic activity that
needs further investigation.
Kinetic Analysis of Mutated Ffase Shows Key Residues for
Activity—Previous work on Sw. occidentalis Ffase points to the
region in the vicinity of Glu-230, the residue acting as acid-base
catalyst in the hydrolytic reaction, as responsible for substrate
recognition and as a potential acceptor subsite for sucrose in
the transfructosylation reaction (8, 15). In fact, the substitution
Q228V caused a large reduction in the enzymatic catalytic effi-
ciency (kcat/Km) on substrates of different size (short, sucrose;
medium, nystose; long, inulin), whereas N254A showed a sig-
nificant reduction on nystose. In addition the substitutions
K181F, S196L, and P232V also reduced this parameter on
sucrose, whereas the two last increased twice the transferase
capacity of the enzyme. Moreover, Ffase crystal structure shows
that residues in this region also participate in an intricate net of
polar interactions with other residues located at the active site
within the dimer interface, which may also be directly involved
in substrate recognition. Therefore, substitutions on these res-
idues may affect/distort the dimer interface and consequently
the enzymatic activity even when they are not directly involved
in substrate binding.
FIGURE 5. Ffase active site specificity. A, stereo pair of the D50A-inulin com-
plex with relevant residues highlighted. Only five fructose units that interact
with Ffase are shown for clarity. Loop 70–80, absent in the A. japonicus FT, is
in ribbon representation. Aromatic residues defining the hydrophobic wall of
the active site are colored in white, and the three catalytic residues are pink.
B, A. japonicus FT complexed with nystose (PDB code: 3LYH). Relevant resi-
dues are represented as sticks. Phe-118 is the unique hydrophobic residue
present in the A. japonicus FT active site, with the fructose unit located at
subsite ϩ3 at an equivalent position to Ffase Trp-76. Two long insertions,
absent in the known GH32 hydrolases, residues 140–153 and 320–331, are
highlighted and hold Ile-143 and Gln-327, two residues proposed to provide a
ϩ2 subsite responsible for the formation of levan- or neo-type products.
C, levansucrase from B. subtilis complexed with raffinose (PDB code: 3BYN).
The Glu-340/Arg-360 pair, participating in subsites ϩ1 and ϩ2, is topologi-
cally and structurally equivalent to the Ffase Gln-228/Asn-254 pair. The inter-
actions observed with the sugar at subsites ϩ1 and ϩ2 are conserved with
levansucrases, and the different product specificities are proposed to be
modulated by a distant loop that is represented in the figure.
With the aim of determining the precise role of the Gln-176,
Gln-228, and Asn-254 positions, we extended the previous
respectively, which is unique to the FT enzyme. Interestingly, mutational study and, in addition to the hydrolytic activity
these two insertions provide Ile-143 and Gln-327, two residues (Table 2), the role of these substitutions on the transferase
involved in binding raffinose and therefore proposed to provide activity (Table 3 and Fig. 6) was evaluated. Gln-176 is involved
a ϩ2 subsite responsible for the formation of levan- or neo-type in substrate recognition at subsite ϩ1 by interaction through a
products in A. japonicus FT (16). An alternative ϩ2 subsite for water molecule, but it also interacts with Arg-178 (in the RDP
the formation of an inulin-type product is defined by the motif). Although the Q176S mutation significantly reduced
A. japonicus FT Tyr-404 and Glu-405, both also involved in (ϳ6-fold) the enzyme catalytic efficiency in nystose, and the
subsite ϩ3 and located near the position of Ffase Trp-314, with Q176N replacement produced a moderately decreased effi-
this last residue conserved in all the microbial enzymes. There- ciency (Յ3-fold) in the three substrates, no clear influence of
fore, it is apparent that none of determinants proposed as this position on the general hydrolytic behavior can be high-
JUNE 1, 2012•VOLUME 287•NUMBER 23
JOURNAL OF BIOLOGICAL CHEMISTRY 19681

Sw. occidentalis ␤-Fructofuranosidase Specificity
lighted (Table 2). Therefore, we suggest that the contribution of the Q228V substitution had a more drastic effect in reducing
Gln-176 to the enzymatic activity might be related to its inter- the production of 1-kestose, whereas the Q228T change
actions with other amino acids. Indeed, the transfructosylating affected largely to that of 6-kestose.
activity of the enzyme was not affected by any of the three
changes made in this position (Fig. 6 and Table 3), indicating
that most likely Gln-176 does not determine the acceptor site
for sucrose in transfructosylation reactions.
With regard to Asn-254, all of the substitutions confirmed
the contribution of this residue in the recognition/hydrolysis of
medium (catalytic efficiency reduced ϳ3–11-fold) and long
size substrates (catalytic efficiency reduced ϳ3–13-fold for
N254D/N254T/N254E). In particular, the N254E replacement
produced a rearrangement of Trp-314 orientation and a subse-
quent change in subsite ϩ3 (Fig. 4), which agrees with the
10-fold decreased efficiency of this mutant toward medium and
long chain substrates. Furthermore, all mutations tested
showed a decrease in the initial Ffase transferase activity at
sucrose up to 0.75 M (Fig. 6). This effect is more evident in the
N254E mutant, which shows about 20% of the transferase activ-
ity even at 1 M sucrose after 24 h of reaction (Table 3 and Fig. 6).
With the exception of the N254E mutant, for which the active
site might be putatively distorted as proposed above, the other
variants produced, at high sucrose concentrations, an amount
of FOS analogous to the wild-type enzyme. However, it is note-
worthy that most substitutions at this position seemed to mod-
ify the product specificity, with Ffase mutants able to synthesize
neokestose (Fig. 7). Moreover, short chain amino acids, such as
Thr or Ala, produced neokestose to a higher extent than 1-kes-
tose, which may well indicate the relevance of this position in
product specificity.
On the contrary, some Gln-228 substitutions had a greater
effect on Ffase hydrolytic activity (Table 2). Thus, the presence
of short side chain amino acids in this position (Thr and Val)
clearly reduced the catalytic efficiency of the enzyme toward
long (inulin, ϳ10–20-fold) and medium size substrates (nys-
tose, ϳ102-fold), whereas replacement by similar size residues
(Asn or Glu) had little effect on this parameter using medium
(ϳ1.5–2-fold decreased) and long size substrates (ϳ1.3–3-fold
increase); this suggests that the length of amino acid chain at
position 228 is essential for its hydrolytic activity. Moreover,
the transfructosylation assay proved that Gln-228 has to be
involved in the transfructosylating reaction mechanism, as its
replacement by Val or Glu considerably reduced the initial rate
at sucrose concentrations in the 0.25–1 M range (Fig. 6). On the
contrary, replacement by Thr and Asn increased (ϳ1.5-fold)
the initial transfructosylating ability at 1 M sucrose, thereby sug-
gesting that polar residues smaller than glutamine might allow
a better recognition or positioning of the transferred sucrose.
However, as seen in Table 3, none of the four substitutions
analyzed was able to improve the total amount of FOS pro-
duced after 24 h of reactions. Interestingly, the specificity of the
product is affected by the nature of the replacement, and thus
In conclusion, it is clear that both, Gln-228 and Asn-254,
configure the acceptor substrate binding site for transfructosy-
lation reactions in Ffase and appear to be responsible for the
inulin/levan-type FOS specificity of this enzyme.
Variants of Ffase Show Redesigned Activity—Our mutagene-
sis analysis gave valuable insights into the GH-J clan enzymes
and delineated an interesting region for further studies in pro-
tein engineering to obtain tailor-made products. In fact, we
found two variants of the enzyme with clearly redesigned func-
tional features. First, as shown in Table 3 and Figs. 6 and 7, the
Q228V mutant yields a 30% decrease in the total amount of FOS
produced (ϳ30 versus 43 g/liter), but the product is almost
specifically 6-kestose. On the other hand, the N254T mutant
produces a similar amount of FOS as the native enzyme in these
conditions, but these FOS are of a more varied type, with
neokestose produced in similar quantity to 1-kestose. There-
fore, this is an excellent example of an efficient way of manip-
ulating the specificity of the recovered FOS, which may be
TABLE 3
FOS produced by Ffase mutants after 24-h reactions
Enzymes (1 unit mlϪ1) and 1 M sucrose were incubated for 24 h. FOS is indicated as
g literϪ1. ND, not detected.
Ffase
Total
FOS
6-K/1-K
ratio
mutant
Neokestose
1-Kestose
6-Kestose
WT
ND
ND
ND
ND
0.5
9.5
5.3
6.6
6.1
0.8
2.6
2.1
2.1
1.3
8.6
3.6
4.1
33.4
21.1
24.7
18.9
28.7
3.3
42.9
26.4
31.3
25.0
30.0
6.9
3.5
Q176S
Q176N
Q176E
Q228V
Q228T
Q228N
Q228E
N254A
N254T
N254D
N2254E
3.9
3.7
3.1
35.9
1.3
1.0
0.8
24.6
23.2
13.5
28.4
12.5
7.3
27.6
25.3
16.5
48.8
18.0
11.4
11.7
11.0
10.4
3.3
ND
1.7
11.8
1.9
3.5
ND
1.8
FIGURE 6. Effect of sucrose concentration on Ffase mutant transferase activity. Enzymes (1 unit mlϪ1) and sucrose (250–1000 mM) were incubated for 3 h.
The amount of carbohydrates formed or hydrolyzed/min was quantified by HPLC in the 1–3 h interval (initial velocity conditions). 6-Kestose was the only
transfructosylation product detected in all reaction mixtures, and the amount of sucrose hydrolyzed was lower than 10% (w/w) in all cases.
19682 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 23•JUNE 1, 2012

Sw. occidentalis ␤-Fructofuranosidase Specificity
FIGURE 7. FOS profile produced by Ffase mutants. Frame of HPLC chromatograms (10.5–15 min) obtained using enzymatic variants with new product
specificity. Enzymes (1 unit mlϪ1) and 1 M sucrose were incubated for 24 h.
adjusted to the requirements in the pursued prebiotic features, within the dimer, not only through their side chain, as is the case of
i.e. a specific versus a broader spectrum product.
Gln-435, but also through the Ser-434 main chain. This explains
the higher hydrolytic activity observed against nystose than
sucrose and gives the first experimental evidence of the direct role
of a GH32 supplementary ␤-sandwich domain in substrate bind-
ing. Furthermore, this restricted architecture of the active site
seems to be adapted to the polymeric conformation of inulin and
to induce a fixed conformation of bound fructooligosaccharides.
The complexes described here also assign a specific role to
the previously identified key residues, Gln-228 and Asn-254, in
shaping subsites ϩ1 and ϩ2 for donor substrate binding. Thus,
Gln-228 is linked to fructose located at subsite ϩ1 through its
O3 atom, whereas Asn-254 is linked to the O3 of fructose
located at ϩ2, and both residues are involved in oligosaccharide
binding of both fructose units through water molecules. Fur-
thermore, on the basis of these experimental complexes, the
binding mode of putative transfructosylating product com-
plexes can be modeled. This allows us to infer how the acceptor
sucrose would be positioned in subsites ϩ1 and ϩ2 of an
enzyme-fructose intermediate complex for the subsequent
transfer of this sugar with the formation of a ␤(2–1) or ␤(2–6)
linkage to produce both 1-kestose and 6-kestose. This model
illustrates how both residues may be involved in the transfruc-
tosylating activity of the enzyme, and certainly, their implied
involvement in both H/T activity and specificity has been
proved by mutagenesis analysis. According to the activity
observed in the mutated proteins, the Gln-228 would be essen-
tially responsible for conferring affinity for acceptor sucrose,
whereas Asn-254 seems to be most responsible for product
specificity. Furthermore, we have generated two variants of
Ffase that show redesigned transfuctosylating ability; thus, the
Q228V mutant gives almost specifically 6-kestose, whereas the
N254T mutant yields a broad spectrum FOS product, adding
neokestose to the 6-kestose/1-kestose mix produced by the
wild-type enzyme.
Proposed Transfructosylating Mechanism—On the basis of
the experimental complexes described here, and by superimpo-
sition with other reported complexes, we have modeled the
putative 1-kestose and 6-kestose products into the Ffase active
site (Fig. 8A). As observed, Gln-228 could be linked to the fruc-
tose accommodated at subsite ϩ1 of both trisaccharides,
whereas Asn-254 seems able to link the glucose of 6-kestose but
is too far from the position inferred for glucose in 1-kestose,
which in turn would stack against the Trp-76 side chain. These
aspects are most relevant when taking into account that the
binding mode of the product complexes at subsites ϩ1 and ϩ2
illustrates how the acceptor sucrose would be positioned in the
catalytic site of a putative enzyme-fructose intermediate com-
plex for the subsequent transfer of this sugar with the formation
of ␤(2–1) or ␤(2–6) linkage to produce 1-kestose and 6-kes-
tose, respectively (Fig. 8B). Therefore, it seems that the binding
mode of the acceptor sucrose linked to Gln-228 and Asn-254 is
preferred by Ffase, with the concomitant production of a 3-fold
amount of 6-kestose/1-kestose. Furthermore, this model is
consistent with the Q228V mutant having a decreased yield of
FOS and an almost exclusive production of 6-kestose, as this
mutant showed a reduced affinity against sucrose but still kept
a polar link to Asn-254 only in this binding mode, consequently
selecting 6-kestose as main product. On the other hand, the
N254T substitution may conform to a larger subsite that allows
a broader range of orientations to accommodate the acceptor
sucrose and therefore also be able to generate neokestose effi-
ciently (Fig. 8B).
DISCUSSION
We proposed previously that, unlike other known GH32 hydro-
lases, Sw. occidentalis Ffase presents a unique active site putatively
able to recognize medium and long chain fructooligosaccharides.
The structure of its complexes with fructosylnystose and inulin
described here presents a detailed picture of the extended net of
interactions that keeps the substrate tightly bound at the active
site. Accordingly, the enzyme presents up to five subsites for sub-
strate binding (Ϫ1toϩ4), and what is more outstanding, the sugar
unit located at subsite ϩ3 is recognized by direct interaction with
As mentioned above, the Gln-228/Asn-254 Ffase residues
are structurally equivalent to Asp-239/Lys-242 of A. thaliana
invertase, which is reported to accomplish a crucial role in
sucrose binding and stabilization. In fact, no structural equiva-
lents are found in FEH, and this feature is proposed to confer
residue from the ␤-sandwich domain of the adjacent subunit invertase/FEH specificity in plant enzymes (14). In agreement
JUNE 1, 2012•VOLUME 287•NUMBER 23 JOURNAL OF BIOLOGICAL CHEMISTRY 19683

Sw. occidentalis ␤-Fructofuranosidase Specificity
FIGURE 8. Proposed mechanism of Ffase transfructosylating activity. A, the inferred position of the product 1-kestose (green) at the Ffase active site was
obtained by superimposition of the C. intybus FEH 1-kestose coordinates (PDB code: 2AEZ) onto the fructose at the Ϫ1 subsite of the experimental Ffase-
substrate complexes. The position of the product 6-kestose (pink) is inferred from superposition of its coordinates extracted from the CSD (Refcode CELGIC)
onto the fructose found at subsite Ϫ1. The position of the Fru-Glu portion in both complexes represents a potential binding mode of acceptor sucrose for the
formation of ␤(2–1) or ␤(2–6) linkage. B, schematic illustration of the proposed mechanism. The donor substrate sucrose is hydrolyzed by nucleophilic attack
forming the fructosyl-Ffase intermediate. A subsequent sucrose molecule (the acceptor substrate) can enter the active site pocket with binding mode 1
generating 1-kestose or, alternatively, with binding mode 2 generating 6-kestose. Additionally, the Q228V/Q228T/Q228N and the N254T/N254D/N254A Ffase
mutants are able to produce neokestose.
with this role, the Aspergillus niger exoinulinase presents Val- Q228T mutations (Thr is found in C. intybus FEH) abolish the
239/Asn-265 at these positions (42). However, the mutagenesis hydrolysis of long substrates while keeping moderate invertase
analysis of Ffase shows an opposite trend, and the Q228V and activity. Moreover, this pair is equivalent to the A. japonicus FT
19684 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 23•JUNE 1, 2012

Sw. occidentalis ␤-Fructofuranosidase Specificity
Glu-318/His-332 (16), where it is involved in shaping subsite regulates specificity within GH32 and GH68 enzymes, where
ϩ1 (Fig. 5B), and also to the Asp-244/Gln-247 pair of the FT the structurally equivalent pair of residues seems to be a poten-
(6-SST/6-SFT) from P. terminalis that has been reported tial target for the rational design of superior enzymes showing
recently to be involved in both donor and acceptor substrate redesigned activities. This is an issue of utmost biotechnologi-
binding (41). In fact, the uniqueness of a Gln in this Asp/Gln cal interest for generating tailor-made FOS directed, ideally, to
pair has been related to the wide range of acceptor specificities the manipulation of the gut microbial communities (47) with an
and branching capacities of this plant FT. Consequently it is associated benefit to human health (48).
clear that all of the structural work carried out on GH32
Acknowledgments—We thank the staff of the European Synchrotron
Radiation Facility for providing beam time and technical assistance
at beamlines ID23-2 and ID14-1 and also the Fundación Ramon
Areces for the institutional grant to the Centro de Biología Molecular
Severo Ochoa. We also acknowledge Asunción Martin for excellent
technical assistance.
enzymes assigns a crucial role to the corresponding equivalent
couple in modulating the distinctive features particular to each
enzyme, but Ffase shows a unique Gln substituting for the
acidic residue within this pair, which may be related to its
hydrolytic activity against both long and short substrates.
Accordingly, the structurally equivalent pair of the GH68
family (Fig. 5C) stabilizes the glucose part of sucrose in levan-
sucrases (Glu-340/Arg-360 in Bacillus subtilis levansucrase),
and it has been shown by mutagenesis that changing Arg
altered the polymerization degree of the product and the trans-
fructosylation/hydrolysis ratio but not the linkage type of the
products, i.e. it does not modulate the product specificity (43).
Moreover, it has been shown recently (17) that both residues
are present in GH68 levansucrases and inulosacarases, the
product linkage type being determined by non-conserved resi-
dues in the more distant ϩ3 and ϩ4 subsites. Interestingly, and
unlike the other GH32 enzymes, the corresponding pair of
Ffase Gln-228/Asn-254 is not only structurally but also topo-
logically equivalent to that in levansucrases and inulosucrases.
However, in this case the involvement of both residues in the
activity is different, as both show a direct role in altering the
ratio H/T, but Asn-254 seems also to be involved in modulating
the specificity of the transfructosylating product.
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19686 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 23•JUNE 1, 2012

Protein Structure and Folding:
Structural and Kinetic Insights Reveal
That the Amino Acid Pair Gln-228/Asn-254
Modulates the Transfructosylating
Specificity of Schwanniomyces occidentalis
β-Fructofuranosidase, an Enzyme That
Produces Prebiotics
Miguel Álvaro-Benito, M. Angela Sainz-Polo,
David González-Pérez, Beatriz González,
Francisco J. Plou, María Fernández-Lobato
and Julia Sanz-Aparicio
J. Biol. Chem. 2012, 287:19674-19686.
doi: 10.1074/jbc.M112.355503 originally published online April 16, 2012
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