1 M. Angela Sainz-Poloa*, Mercedes Ramírez-Escuderoa*, Alvaro Lafrayab& , Beatriz Gonzáleza ...

25 Pages·2013·1.07 MB·English

by  Julia Sanz Aparicio

JBC Papers in Press. Published on February 21, 2013 as Manuscript M112.446435
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M112.446435
Structure of Saccharomyces invertase

THE THREE-DIMENSIONAL STRUCTURE OF SACCHAROMYCES INVERTASE: ROLE OF A NON-
CATALYTIC DOMAIN IN OLIGOMERIZATION AND SUBSTRATE SPECIFICITY

M. Angela Sainz-Poloa*, Mercedes Ramírez-Escuderoa*, Alvaro Lafrayab&, Beatriz Gonzáleza, Julia
Marín-Navarrob, Julio Polainab and Julia Sanz-Aparicioa#
aDepartamento de Cristalografía y Biología Estructural, Instituto de Química-Física "Rocasolano",
CSIC, Serrano 119, 28006-Madrid, Spain.
bInstituto de Agroquímica y Tecnología de Alimentos, CSIC, Paterna, Valencia, Spain.

Running title: Structure of Saccharomyces invertase
* Contributed equally
& Present address : Instituto de Catálisis y Petroleoquímica, CSIC, Madrid, Spain. D
o
# All correspondence should be addressed to: J. Sanz-Aparicio. Tel: [phone]. Fax: [phone] w
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2431. e-mail: [email protected] a
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fro
Keywords: Invertase Saccharomyces, Michaelis-Menten,
  Glycoside hydrolase family 32, X-ray m
h
crystallography, Biotechnology, prebiotics. ttp
Saccharomyces invertase at 3.3 Å resolution ://w
w
Background: Invertase is a fundamental enzyme showing that the enzyme folds into the catalytic w
for sugar metabolism in yeast and a classical β-propeller and β-sandwich domains .jbc.o
characteristic of GH32 enzymes. However, rg
model in early biochemical studies. b/
Saccharomyces invertase displays an unusual y
Results: Invertase shows an unusual octameric gu
quaternary structure. Monomers associate in e
s
quaternary structure composed of two types of two different kinds of dimers that are in turn t o
n
dimers. assembled into an octamer, best described as a Ja
n
u
Conclusion: A peculiar pattern of monomer tetramer of dimers. Dimerization plays a ary
assembly through non-catalytic domain determinant role in substrate specificity 2, 2
interactions determines invertase specificity. because this assembly sets steric constrains that 019
limit the access to the active site of the
Significance: Unravelling the structural features
disaccharide (sucrose) or short oligosaccharides
that rule enzyme modularity casts new light to
of up to four units. Comparative analysis of
understand protein-carbohydrate recognition.
  GH32 enzymes shows that formation of the
Saccharomyces invertase octamer occurs
through a β-sheet extension that seems unique
SUMMARY
to this enzyme. Interaction between dimers is
determined by a short amino acid sequence at
Invertase is an enzyme widely distributed
the beginning of the β-sandwich domain. Our
among plants and microorganisms that
results highlight the role of the non-catalytic
catalyzes the hydrolysis of the disaccharide
domain in fine-tuning substrate specificity and
sucrose into glucose and fructose. Despite the
thus supplement our knowledge on the activity
important physiological role of Saccharomyces
of this important family of enzymes. This, in
invertase and the historical relevance of this
turn, gives a deeper insight into the structural
enzyme as a model in early biochemical studies,
features that rule modularity and protein-
its structure had not yet been solved. We report
carbohydrate recognition.
here the crystal structure of recombinant

  1

Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure of Saccharomyces invertase
INTRODUCTION mechanism in which an aspartate located close to
the N-terminus acts as the catalytic nucleophile
Invertase (EC3.2.1.26; β-fructofuranosidase) and a glutamate acts as the general acid/base
catalyzes the hydrolysis of the disaccharide catalyst. The reaction proceeds through attachment
sucrose (table sugar) into glucose and fructose, of the aspartate nucleophile to a fructosyl unit of
being a major enzyme present in plants and the donor substrate. The fructosyl is subsequently
microorganisms. Since the yeast Saccharomyces released by hydrolysis or transferred to an acceptor
was one of the preferred materials in early sugar substrate (transglycosylation). In recent
biochemical studies, yeast invertase became one of years, the crystallographic structure of several
the classical model enzymes for studies on protein GH32 from bacteria (14, 15, 16) and eukaryotes
synthesis and activity, and the excretion of (17, 18, 19, 20, 21) have been reported. A close
glycoproteins. Mitscherlich described in 1842 the phylogenetic relative to Saccharomyces invertase
existence in yeast of a substance capable of is an inulinase from Schwanniomyces occidentalis
inverting dextrorotatory cane sugar into a (22). The characteristic structural feature of GH32
levorotatory sugar that was identified in 1847 by enzymes, shared by GH68 enzymes included in the
Dubrunfaut as a mixture of glucose and fructose. same GH-J clan, is a 5-fold β-propeller catalytic
In 1860, for the first time, Berthelot carried out the domain consisting of five blades, each one
isolation of invertase (1). At that time, the whole composed of four antiparallel β-strands with ‘W’ D
o
theory of enzyme kinetics was based on topology, which surround a central negatively w
n
experimental results obtained with yeast invertase charged active site cavity. The GH32 differs from loa
d
(a2ss).o cSiaintecde tion vtehreta syee awsta sc eflolsu nadn dt oi tsb ep uinritfiimcaattieolny dthoem aGinH a6p8p efnadmeidly t ob tyh e acna taaldydtiict idoonmal aiβn-. sandwich ed from
required the preparation of yeast extracts, it was Besides its historic importance in the http
considered to be an intracellular enzyme. development of Biochemistry, Saccharomyces ://w
However, de la Fuente and Sols (3) showed that invertase has extensive industrial applications. It is ww
the enzyme is secreted by the yeast cells and that one of the most widely used enzymes in .jb
c
the hydrolysis of sucrose is extracellular. confectionary to make liquid centres in candy .org
Subsequently, it was discovered that the yeast making, and is also used during fermentation of by/
produces two types of invertase: a heavily cane molasses into ethanol. A new potential gu
e
glycosylated secreted form and a non-glycosylated application is the synthesis of prebiotic fructo- st o
n
intracellular one (4, 5). Yeast invertase is encoded oligosaccharides used in functional foods and Ja
n
by a family of repeated SUC genes (6, 7, 8, 9). The pharmaceutical formulations (23). The use of ua
enzyme is normally secreted by yeast as a heavily prebiotics to orchestrate the gut microbiota ry 2
glycosylated octameric protein. The large mass of composition, with the associated benefit to human , 20
1
the protein leads to it being trapped in the cell wall health, is an emerging issue of the utmost 9
(10, 11). Both, the non-glycosylated, biotechnological interest (24)
cytoplasmatic and the secreted form of invertase, In this communication we report the three-
are encoded by the same gene (8). These two dimensional structure of the Saccharomyces
forms are transcribed as two mRNAs of different invertase, produced in Escherichia coli, by
length, which are translated into polypeptides of expression of the SUC2 coding sequence without
different size. The longer one, in addition, encodes the 5' end of the gene corresponding to the
the signal peptide needed for secretion (12). secretion signal peptide. Our results reveal the
structural basis of the unique oligomerization
On the basis of sequence similarity, invertase pattern observed in SInv, and provide key factors
is classified within family 32 of glycoside to understanding the enzymatic activity and
hydrolases (13). In addition to invertases, this specificity of this important enzyme.
family (designated GH32) includes inulinases and
levanases involved in the hydrolysis of fructose-
containing polysaccharides, and also EXPERIMENTAL PROCEDURES
transglycosylases with fructose transferase Cloning, expression and purification- The
activity. GH32 enzymes act by a retaining gene encoding SInv was expressed in Escherichia
2
Structure of Saccharomyces invertase

coli Rosetta2 cells carrying plasmid SUC2-pQE. Å resolution range and a Patterson radius of 45 Å,
The resulting His-tagged protein was purified by which after rigid body fitting led to an R factor of
nickel-affinity chromatography, as previously 46%. Crystallographic refinement was performed
described (23). For crystallization, the eluted using the program Refmac (29) within the CCP4
fractions containing Suc2 (>95% purity according suite with flat bulk-solvent correction, and using
to SDS-PAGE analysis and Coomassie-blue maximum likelihood target features. Tight local
staining) were dialyzed (1/10000) against 0.05 M non-crystallographic symmetry (NCS) and jelly
Tris-HCl buffer pH 7 and concentrated using 20K body restraints were applied during first steps of
cutoff membrane ultrafiltration (Pierce). For refinement. Free R-factor was calculated using a
activity assays, SInv was dialyzed (1/10000) subset of 5% randomly selected structure-factor
against 0.05 M phosphate buffer pH 7, 150 mM amplitudes that were excluded from automated
NaCl. refinement. Some regions of the polypeptide chain,
Crystallization and data collection- in particular loops 190-200 and 230-240, and the
Crystallization of SInv (10 mg mL-1 in 0.05 M β-strand 228-250, all located at the interface
Tris-HCl, pH 7) was performed on Cryschem between dimers, were not visible in the electron
(Hampton Research) sitting drop plates at 18 ºC. density of molecules EFGH and were excluded
Small bars grew in 3% Peg 3350, 5% MPD, 0.6 M from the model during the first stages of the
D
magnesium formate, 1 mM TCEP, 0.33 M refinement. Furthermore, two NCS groups were ow
n
guanidinium chloride, 0.1 bis-tris pH 6.5 (drop defined composed of molecules ABCD and lo
a
ratio protein/precipitant 2:1) by vapour diffusion at EFGH, respectively. After iterative refinement and de
d
room temperature. The crystals diffracted to 3.3 Å rebuilding of these regions using the program fro
m
resolution. Many attempts to improve resolution at COOT (30), the final 2Fo-Fc map showed h
low temperature, or using other techniques as continuous density for the whole protein. At the ttp://w
streak and micro seeding or using agarose (2-3 %) latter stages, water molecules were included in the w
w
in the crystallization drops were unsuccessful. For model, which, combined with more rounds of .jb
more details about crystallization behaviour of this restrained refinement, led to a final R-factor of c.o
rg
SInv see Sainz-Polo et al (25) 22.9 (Rfree = 23.7) for all data set up to 3.3 Å b/
y
Crystals of SInv belonged to P3121
  space resolution. Refinement parameters are reported in gu
group with eight molecules in the asymmetric unit Table 1. es
t o
and 75% solvent content within the unit cell. For Stereochemistry of the models was checked n J
a
data collection, native crystals were transferred to with PROCHECK (31) and MOLPROBITY (32). nu
a
cryoprotectant solutions consisting of mother The figures were generated with PyMOL (33). ry
2
liquor plus 25% (v/v) glycerol before being cooled Analysis of the interfacial surfaces and the , 2
0
to 100 K in liquid nitrogen. Diffraction data were oligomer stability was done with the Protein 19
collected using synchrotron radiation at the Interfaces, Surfaces and Assemblies service
European Synchrotron Radiation Facility (ESRF, (PISA) at the European Bioinformatics Institute
Grenoble) on the ID23.1 beamline. Diffraction (34). RMS deviation analysis used the program
images were processed with iMOSFLM (26) and SUPERPOSE within the CCP4 package (27)
merged using the CCP4 package (27). A summary Activity assays- Purified SInv was incubated
of data collection and data reduction statistics is for different times with sucrose, 1-kestose,
shown in Table 1. nystose, raffinose or inulin (from dahlia tubers) in
Structure solution and refinement- The 100 mM acetate buffer, pH 4.8 at 50 ºC. The
structure of SInv was solved by molecular enzyme was inactivated by heating at 95 ºC for 10
replacement using the MOLREP program (27). minutes. The calculation of the initial velocity of
The structure of SoFfase (PDB code 3KF3) was hydrolysis was based on the kinetics of product
used to prepare the search model using the release. The products of the reaction were
program Chainsaw (28) and a protein sequence analyzed by anion-exchange chromatography
alignment of SInv onto SoFfase. A solution through a CarboPac PA-100 column (4x250 mm),
containing eight molecules in the asymmetric unit coupled to a pulsed amperometric detector
(A-H) was found using reflections within 50 – 3.5 (Dionex) as previously described (23).

  3
Structure of Saccharomyces invertase
EF/GH, which are located at opposite vertices of
the square. The subunits of these two classes of
RESULTS dimers associate differently with each other and,
The invertase of the yeast Saccharomyces thus, dimers EF/GH can be described as an “open”
(SInv) has been produced in E. coli, purified and assembly whereas dimers AB/CD form a “closed”
crystallized, as previously reported (25). We arrangement. Structural superposition of chain B
present here the three-dimensional structure of the to chain F shows that it would be necessary a 15°
enzyme solved by molecular replacement at 3.3 Å rotation to bring monomer A into E position
resolution. Experimental and structure (Figure 1c), three regions of the SInv monomer
determination details are given in Materials and acting as hinges (coloured blue in Figure 1d, left).
Methods and in Table 1. The crystals belong to the On the other hand, and likewise the known
P3 21
 space group, the asymmetric unit containing GH32 members, SInv subunit folds into two
1
a complete homo-oligomer of eight subunits. Each domains, a catalytic β-propeller domain (residues
chain (A-B-C-D-E-F-G-H) consists of 512 1-334) and a β-sandwich domain (residues 342-
residues with a molecular mass of 58.5 kDa as 512), linked by a short loop (Figure 1d, right). The
calculated from its primary structure. The β-propeller domain, in turn, is assembled from five
imposition of tight non-crystallographic symmetry
blades (I-V) each consisting of four antiparallel β-
(NCS) during early refinement led to a model with D
strands (A to D, from the axis towards the outside o
eight identical subunits. Most of the polypeptide w
chain exhibited good electron density but there of the propeller) connected by turns in a classical nloa
“W” pattern. Blade I is the most, and blade IV the d
e
were some segments that remained undefined, d
mainly the regions located at the subunits lseitses icso nlosecravteedd aamt tohneg aGxiHs 3o2f ftahme ilpyr.o Tpehlele cr aatanldy tiics from
interface. Consequently, and according to the shaped by the loops connecting the different http
oligomerization pattern shown by SInv (described blades (L1-L4) and the turns linking strand B to C ://w
below), two strict NCS-groups were defined so w
within each blade (TI-TV). On the other hand, the w
that chains A-B-C-D and E-F-G-H were refined .jb
ienledcetproennd denentlsyi.t yT ihni sa lsl cthheem seu bleudn ittso, wa hceoren tianllu othues ββ--sshheeeett s dfoomldaeidn inhtaos at wβ-os asnidxw-sitcrahn tdoepdo laongtyip, aarnadll eisl bc.org/
residues could be fitted. The final model showed a the region presenting the lowest sequence y gu
homology among GH32. es
root mean-square (RMS) deviation of 1.02 Å t o
The quaternary structure of SInv in solution n
between respective Cα atoms from both NCS- has been analyzed by different methods. Jan
groups, the differences being restricted to some ua
Recombinant SInv subjected to gel-filtration ry
particular regions of the polypeptide chain, as it 2
chromatography eluted mainly as a peak , 2
will be explained below. 0
corresponding to the size of an octamer, although a 1
9
Crystallized SInv is an octameric enzyme-
small fraction of the protein appeared as
The molecular weight of purified SInv, 428 kDa
aggregates of higher molecular mass (Figure 2a).
(Figure 2b), was consistent with an octameric
This tendency to aggregate was also observed in
association as proposed before (25). The structural
non-denaturing PAGE gels (Figure 2b). Oligomers
analysis sowed that it is a flat square-shaped
of less than eight units (hexamers, tetramers and
octamer with dimensions 130 x 130 x 110 Å that is
dimers) were also detected and the incubation of
made-up of eight subunits related by non-
the enzyme at 47 ºC for 1-2 hours, or treatment
crystallographic two-fold symmetry parallel to the
with 2 M urea, stimulated the dissociation of the
c-axis, in a 222 arrangement (Figure 1a). Its
octameric form. The enzyme was active in all
molecular surface is 136,914 Å2, with a total
these different oligomerization states, as seen by
buried surface area of 22,296 Å2.
the zymogram test (Figure 2b). Variability in
However, this octamer is best described as a
quaternary structure was also observed by
tetramer of dimers that oligomerize by inter-
ultracentrifuge analysis (not shown), which
subunit extension of the two β-sheets that end the
indicated that SInv was octameric but showed a
β-sandwich domain within each subunit (Figure
decrease in the average molecular mass at the
1b). Furthermore, close inspection reveals that
highest centrifugation force (11,000 rpm.). All
SInv forms two class es of dimers, AB/CD and
4
Structure of Saccharomyces invertase

these results are well in agreement with those other proteins (36). These intermolecular
reported for both intracellular and secreted forms interactions between the hydrogen-bonding edges
of the native enzyme (10, 35). of β-sheets is considered a fundamental form of
Different atomic interactions define dimer biomolecular recognition (like DNA base pairing)
association- Table 2 gives the polar interactions and is involved not only in oligomerization and
found within each interface of the octamer. First, protein-protein interactions, but also in protein
dimers AB/CD are tightly associated by aggregation, as it occurs in β-amyloid fibrils
interactions among both, their catalytic and β- formation (37).
sandwich domains (Figure 1c). Fourteen of a total Table 3 summarizes the main features of the
of 32 hydrogen bonds are made between their different interfaces found in the octamer as
catalytic domains, mainly through loop L3 and analyzed by the Pisa program (34). As it is seen in
strand D4, and also through a long loop connecting the table, the interface area of the “closed” dimers
strands C5-D5 (residues 316-328) that makes is twice that found in the “open” dimers, as it is the
many interactions near the catalytic pocket, as it is number of polar interactions. The binding energy
shown in Figure 3a. Furthermore, the catalytic (ΔiG) is negative in all interfaces indicating their
pocket of one monomer is surrounded by loops hydrophobic nature but the P value reveals that the
from the β-sandwich domain of the other, and interfaces AB/CD, with lowest ΔiG, have a large
D
there is a polar interaction from Ser439 to Gly235 hydrophobicity at a higher confidence level. ow
n
located at TIV that stabilizes the dimer interface at Therefore, only the “closed” dimers, AB/CD, lo
a
d
this region. The base of the catalytic pocket is might be expected to exist in solution. The other ed
additionally lined by hydrophobic interactions interfaces may represent weak interactions (38) fro
m
through Phe388 and Phe296 (TV) and direct polar existing in higher oligomers of SInv depending on h
interaction between both β-sandwich domains external conditions. This agrees with the ttp://w
(Table 2). oligomerization behaviour of both cytosolic and w
w
By contrast, the dimers EF/GH lack atomic secreted SInv isoforms (10, 35). .jb
c
interactions between their catalytic domains as it is A few changes in its sequence determine the .o
rg
shown in Table 2, and only keep some hydrogen oligomerization pattern of SInv- The specific role b/
y
links between their β-sandwich domains, half of of the β-sandwich domain in GH32 enzymes gu
e
them not being conserved with respect to the functionality remained elusive for a long time. The st o
“closed” dimers. However, the catalytic pocket is first experimental evidence of its implication in n J
a
also strengthened by a new salt bridge formed dimerization and substrate binding became nu
a
between Asp45 (at loop TI) and Lys385 (at the available when the first structure from a yeast ry 2
loop linking β3−β4 of the β-sandwich domain), enzyme, the Swanniomyces occidentalis β- , 20
1
which encloses a well-defined cavity (Figure 3b). fructofuranosidase (SoFfase), was reported (22). 9
This different pattern of interaction between the Very recently, the unique role of its β-sandwich
subunits of the two kinds of dimer has two direct domain in substrate recognition has been further
implications. Firstly, the EF/GH active site has a demonstrated from the structure of two complexes
wider pocket (Figure 1c). Secondly the lack of with long substrates (39). Although SoFfase and
interactions between the catalytic domains SInv share 68% sequence homology (being 48 %
produces a rearrangement in the dimer interface identical), SoFfase is a dimeric enzyme and higher
regions that leads to the structural differences of aggregation forms have not been detected.
the two kinds of dimers (Figure 1d). Structural superimposition of SInv and
Both types of dimers assemble through a SoFfase (Figure 5) reveal that the catalytic
similar interface that involves the extension of the domains are almost identical and that most regions
two β-sheets of each β-sandwich domain (Figure of the β-sandwich domain are very similar.
4) centred on β1 and β2, respectively. It is Accordingly, the hydrogen link pattern in SoFfase
interesting that the sheet constituting the “inner” dimer is very similar to that of the “closed” SInv
part of the octamer (Figure 4a) forms a regular dimers AB/CD. However, it is remarkable that
antiparallel intermolecular β-sheet, similarly to none of the residues that provide the polar links for
that reported in multimeric lectins, cytokines and the β-sheet extension through β1 and β2 are

  5
Structure of Saccharomyces invertase
conserved, in SoFfase. Consequently, this region substrate, followed by the trisaccharides raffinose
shows poor structural alignment (Figure 5). and 1-kestose, while the tetrasaccharide nystose
Furthermore, the β1-β2 region in SoFfase shows was hydrolyzed at much lower rate (Table 4). SInv
high content in Lys residues (9/20), which are showed no significant activity with inulin as the
solvent exposed at the base of the β-sandwich, substrate.
yielding a positively charged surface that would It has been reported that SInv and SoFfase
prevent dimer association along this region due to have some degree of transglycosylating activity,
electrostatic repulsion effects. giving 6-kestose as the main product. This activity
Quaternary structure determines substrate can be enhanced by mutagenesis (23, 39, 41). In
specificity- The active site of SInv is located at the contrast, homologous plant enzymes of the same
interface within each pair of dimers. Because of GH32 family yield mostly 1-kestose. To
the two different ways subunits can form dimers understand the β(2,6) or β(2,1) nature of the
(Figure 1c), the active sites in these dimers may transglycosylation reaction, the binding site on the
have different environments. Thus, dimers AB/CD putative acceptor sucrose and its orientation
form a very narrow pocket of 10 Å x 10 Å that relative to the fructose unit in the covalent
seems unable to accommodate an oligosaccharide intermediate, must be identified. Figure 6d gives a
with more than three or four sugar units (Figure superimposition of SInv and SoFfase catalytic
6a). In contrast, the wider (20 Å x 16 Å) entrance pockets showing the putative position of the D
o
cavity observed in dimers EF/GH might allow products 1-kestose and 6-kestose. The figure wn
longer substrates (Figure 6b), although this could suggests that the nucleophile (Asp22 in SInv loa
d
e
idnivstoolrvteio na o f stihgen pifoiclyasnatc, cheanreirdgee. tically expensive, hnyumdrboeprhinobgi)c weanlvl iorof nthmee pnot cketot g(eTtrhpe4r8 , Pwhieth8 2 atnhde d from
A comparison of the catalytic pockets of Trp291), are coincident in all the three cavities. http
octameric SInv with SoFfase (Figure 6c) reveals Gln201 is well positioned to make polar links to ://w
that the shape and size of SoFfase cavity is more the fructose unit at subsite +1 in both ww
similar to that within the “open” EF/GH Inv transfructosylating products, Asn228 could link to .jb
c
dimers. Nevertheless, and more significant, the the glucose moiety of 6-kestose, while the glucose .org
chemical nature of the residues that form the β- term of 1-kestose would stack with Trp48. An by/
g
sandwich domain surrounding the active center is essential role is thus assigned to Gln201 to u
e
s
quite different in the two enzymes. SoFfase has facilitate the transfructosylating reaction by t o
n
long-chain residues as Arg473, Glu464, Tyr462 binding the acceptor sucrose, while Asn228 would Ja
n
and Tyr468 and notably Gln435, a residue that, crucially determine the product specificity as ua
ry
together with Ser412, makes direct polar links with predicted (23) and also described for SoFfase (39). 2
, 2
the oligosaccharides at subsite +3 (39, In summary, the architecture of the active 0
1
9
nomenclature according to 40). Furthermore, site, as determined by the way the enzyme
Asn401, Asn403 and Asp471 protrude at the monomers are assembled, explains both substrate
entrance of the slot and define the polar boundaries specificity for hydrolysis (invertase vs. inulinase
that make accessible the cavity from the solvent. activity) and product specificity for
Therefore, most of the relevant SoFfase residues transfructosylation. Thus, the dimerization mode
are highly flexible. By contrast, most of the of SInv modulates its hydrolytic activity
corresponding SInv residues are short-chain precluding the recognition of long chain
aminoacids as Ser412, Ser414, Ser415, Thr379, substrates. This is more apparent for the “closed”
Thr380 and Ser447. Consequently, the SoFfase dimers that would be predominant in aggregation
active site seems more flexible to accommodate states lower than the octamer, or in alternative
long-chain substrates than the two catalytic octameric forms composed exclusively of closed
pockets of SInv. This feature may therefore dimers (see discussion). However, the
illustrate the structural basis for the activity of transfructosylating mechanism would be the same
SInv as an invertase whereas SoFfase is in fact an in both enzymes.
inulinase. As observed when comparing the
activity of SInv with different oligosaccharides,
the highest efficiency was with sucrose as a
6
Structure of Saccharomyces invertase

DISCUSSION soluble protein, is encoded by the same gene as the
The yeast Saccharomyces plays an secreted form (12). Secondary and tertiary
outstanding role in human civilization as the structures of both forms are virtually identical, as
fermentative agent that produces bread, wine and revealed by CD spectroscopy analysis (35). These
beer. Saccharomyces owes its predominant considerations led us to carry out heterologous
position as a fermentative microorganism to a very expression of SInv in E. coli, which yielded
successful metabolic strategy. In the sugar-rich suitable protein material for crystallization
ecological niches where it dwells, Saccahromyces analysis (25).
performs a highly efficient mobilization of sugars, The analysis of the crystal structure at 3.3 Å
which can be channeled to the production of an resolution that we report here shows that SInv
antiseptic substance (ethanol) that avoids the folds into the catalytic β-propeller and β-sandwich
proliferation of competing microorganisms instead domains characteristic of GH32 enzymes. A dimer
of being used for the production of biomass. The association that shapes the active site has been
function of a set of enzymes involved in sugar found, similarly to that described in the
metabolism, including invertase, is critical for the phylogenetically close β−fructosidase from
prevalence of Saccharomyces in its natural habitat. Schwanniomyces occidentalis (22). However, in
In spite of the important physiological role of the contrast, SInv displays a special assembly of
D
Saccharomyces invertase and its historical dimers into octamers through extension of each ow
n
relevance as a model enzyme in early biochemical subunit β-sandwich domain. The particular lo
a
studies (2) its crystallographic structure had not geometry of the octamer generates “closed” and ded
yet been reported. Although it is secreted by the “open” dimers that are located alternatively at the fro
m
yeast in large amounts, native SInv appears as a vertices of a rectangle. Analysis of the interfaces h
heavy hyperglycosylated protein of heterogeneous and binding energy calculation shows that “closed- ttp://w
molecular mass, which remains trapped in the cell type” dimers are more stable. Although the w
w
wall (10). It is therefore a rather unsuitable octameric form is predominant in a fresh .jb
material for crystallization. The biosynthesis and preparation of the enzyme (Figure 2a), the weaker c.o
rg
secretion of extracellular proteins by eukaryotes is association within the “opened-type” dimer and b/
y
a complex process in which the transit of the the dimer-dimer interface may explain the g
u
nascent polypeptide through the endoplasmic instability manifested by the octamer under est o
reticulum is coupled with core glycosylation and incubation at higher (47 ºC) temperatures (Figure n J
a
protein folding. Hyperglycosylation takes place at 2b) or a high centrifugal force. Consequentially, nu
a
a later stage in the Golgi and does not affect SInv recombinant SInv is predominantly an octamer but ry 2
folding or catalytic activity (42). Due to the fact may exist as a dimer and other oligomeric forms, , 2
0
that core glycosylation is often a necessary as it has been reported for both the cytosolic and 19
condition for proper protein folding, secreted secreted isoforms (10, 11, 35, 43). Furthermore, as
eukaryotic proteins cannot be functionally seen from the structure reported here, SInv
expressed in E. coli. Therefore, the collection of a dimerization plays a determinant role in substrate
sufficient amount of protein for crystallization specificity preventing binding of extended
analysis requires, either, that it is abundantly substrates, which explains its invertase character at
produced by the organism from which derives, or the molecular level.
heterologous expression in a eukaryotic host. Electron micrographs of both internal and
Examples of structurally determined SInv secreted SInv (10, 11) show a similar association
homologues of family GH32 are therefore fungal pattern of spherical units, with different
inulinases abundantly produced by their natural oligomerization states: dimers, tetramers,
host (17, 20, 22), plant fructosylases heterologous hexamers and octamers. Interestingly, secreted
expressed in Pichia pastoris (18, 19) and a plant SInv octamers appear slightly open to one side
fructosyl transferase abundant in leaves (21). while intracellular invertase octamers appear
Saccharomyces produces an intracellular non- mostly as nearly symmetrical closed rectangles,
glycosylated version of the invertase (4). The like the structure presented here. The electron
intracellular version, which is synthesized as a micrographs of secreted invertase octamers

  7
Structure of Saccharomyces invertase
resemble strikingly the model illustrated in Figure catalytic domains with, in principle, unknown
7, in which the protein would be composed of function play a role in fine-tuning enzymatic
“closed” dimers linked by intermolecular β-sheets. function. The contribution of the β-sandwich
While GH32 enzymes are generally highly domain in building the catalytic pocket of GH32
conserved in the β-propeller and less so in the β- yeast enzymes has been previously reported.
sandwich domain, SInv and Schwanniomyces However, a role in higher oligomerization leading
occidentalis fructosidase (SoFfase) are very to new specificity seems unique to SInv. The
similar also in some regions of the β-sandwich production of a complex octameric,
domain, namely the β8-β10 strands and the β6-β7 hyperglycosylated enzyme precludes its diffusion
region. However, strands β1 and β2 that build the outside the periplasmic space. Secretion of
intermolecular β-sheet within the octamer, are less invertase that occurs in many microorganisms
conserved. In fact, homology alignments of yeast represents an evolutionary advantage eliminating a
GH32 sequences show that many of them contain mechanism for sucrose import. The unique quality
a Pro residue at β1 or β2 that probably precludes of Saccharomyces is that it keeps the invertase
the intermolecular β-sheet formation (Figure 8). In trapped on the cell surface avoiding its diffusion in
the medium where it would also aid competing
SoFfase, an unusually large number of solvent
organisms. We hope that the resolution of the
exposed Lys residues are found in this region,
sophisticated molecular architecture of this D
resulting in a positively charged surface that likely o
w
interferes with β-sheet dimerization because of enzyme may contribute to the understanding of nlo
protein-carbohydrate interactions and to the design a
electrostatic repulsion effects. Therefore, the d
e
association of dimers into octamers seems to be a of novel, more efficient enzymes for d fro
biotechnological purposes.
  m
unique SInv feature. h
To conclude, the structure of SInv presented ttp
here is an interesting new example of how non- ://w
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Acknowledgements- We thank the staff of the European Synchrotron Radiation Facility for providing
beam time and for technical assistance at beamline ID23.1. We also thank Simon P. Gough for kindly
reviewing the manuscript.

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