Pc_Cel7D binds to lactose more strongly than cellobiose, while the oppos-ite is true for the homologous Trichoderma reesei cellobiohydrolase Tr_Cel7A.. In the present paper, we report th
Trang 1in complex with product and inhibitors
Wimal Ubhayasekera1, Ine´s G Mun˜oz1,*, Andrea Vasella2, Jerry Sta˚hlberg1
and Sherry L Mowbray1
1 Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden
2 Laboratory of Organic Chemistry, ETH, Ho¨nggerberg, Zu¨rich, Switzerland
Cellulose is the most abundant polymer on earth It
has been estimated that as much as 15% of all
atmo-spheric carbon dioxide is fixed yearly, resulting in vast
quantities of plant biomass, mostly as a complex
mix-ture of cellulose and lignin [1] The recycling of this
carbon is critically dependent on the action of
micro-bial organisms, primarily fungi and bacteria An
understanding of the processes at work is obviously of
enormous environmental importance The enzymes involved are also useful for applications that include, among others, their use in commercial laundry pow-ders, as well as in the de-inking of recycled paper and the synthesis of fine chemicals
Cellulases, the enzymes that hydrolyse cellulose, have been broadly characterized as cellobiohydrolases (1,4-b-d-glucan cellobiohydrolase, EC 3.2.1.91) and
Keywords
Cellulase; cellobiohydrolase; glycoside
hydrolase; Trichoderma reesei;
Phanerochaete chrysosporium
Correspondence
J Sta˚hlberg, Department of Molecular
Biology, Swedish University of Agricultural
Sciences, Biomedical Centre, PO Box590,
SE-751 24 Uppsala, Sweden
Fax: +46 18 536971
Tel: +46 18 471 4566
E-mail: Jerry.Stahlberg@molbio.slu.se
*Present address
Structural Biology and Biocomputing
Programme, Spanish National Cancer Centre
(CNIO), Melchor Ferna´ndez Almagro 3,
28029 Madrid, Spain
(Received 6 December 2004, revised 15
February 2005, accepted 22 February 2005)
doi:10.1111/j.1742-4658.2005.04625.x
The cellobiohydrolase Pc_Cel7D is the major cellulase produced by the white-rot fungus Phanerochaete chrysosporium, constituting 10% of the total secreted protein in liquid culture on cellulose The enzyme is classified into family 7 of the glycoside hydrolases and, like other family members, catalyses cellulose hydrolysis with net retention of the anomeric carbon configuration Previous work described the apo structure of the enzyme Here we investigate the binding of the product, cellobiose, and several inhibitors, i.e lactose, cellobioimidazole, Tris⁄ HCl, calcium and a thio-linked substrate analogue, methyl 4-S-b-cellobiosyl-4-thio-b-cellobioside (GG-S-GG) The three disaccharides bind in the glucosyl-binding subsites +1 and +2, close to the exit of the cellulose-binding tunnel⁄ cleft Pc_Cel7D binds to lactose more strongly than cellobiose, while the oppos-ite is true for the homologous Trichoderma reesei cellobiohydrolase Tr_Cel7A Although both sugars bind Pc_Cel7D in a similar fashion, the different preferences can be explained by varying interactions with nearby loops Cellobioimidazole is bound at a slightly different position, displaced
2 A˚ toward the catalytic centre Thus the Pc_Cel7D complexes provide evidence for two binding modes of the reducing-end cellobiosyl moiety; this conclusion is confirmed by comparison with other available structures The combined results suggest that hydrolysis of the glycosyl-enzyme intermedi-ate may not require the prior release of the cellobiose product from the enzyme Further, the structure obtained in the presence of both GG-S-GG and cellobiose revealed electron density for Tris at the catalytic centre Inhibition experiments confirm that both Tris and calcium are effective inhibitors at the conditions used for crystallization
Abbreviations
GG-S-GG, methyl 4-S-b-cellobiosyl-4-thio-b-cellobioside; IBTG, o-iodo-benzyl-b- D -thio-glucoside; Pc_Cel7D, cellobiohydrolase Cel7D from Phanerochaete chrysosporium; PDB, Protein Data Bank; pNP-Lac, p-nitrophenyl-b- D -lactoside; Tr_Cel7A, cellobiohydrolase Cel7A from Trichoderma reesei.
Trang 2endoglucanases (1,4-b-d-glucan glucanohydrolase,
EC 3.2.1.4) [2] Cellobiohydrolases tend to act
proces-sively from the end of a cellulose chain, that is, they
cleave off a number of cellobiose units in succession
before the enzyme is released [3,4] Endoglucanases cut
cellulose at random positions within the chains, thus
creating new ends from which cellobiohydrolases can
work Efficient degradation of cellulose requires a
synergistic balance between the two types of activities
Cellulases and other glycoside hydrolases have been
classified into structurally related families, based on
sequence homology as well as the patterns of
hydro-phobic residues [5,6] To date nearly 100 glycoside
hydrolase families are defined in the CAZY database
(http://afmb.cnrs-mrs.fr/CAZY/) Efficient
cellulose-degrading fungi generally have at least one member
of glycoside hydrolase family 7 The enzymes in this
family perform hydrolysis with net retention of the
anomeric configuration, in a double-displacement
mechanism through a covalent glycosyl-enzyme
inter-mediate [7,8] Most, but not all, members have a small
cellulose-binding module connected to the catalytic
module by a presumably flexible linker The catalytic
core of this family is a b-sandwich composed of two
large, mainly antiparallel, b-sheets packed onto each
other (Fig 1) A long cellulose-binding site is defined
by loops on one face of the sandwich It has been
demonstrated, for this and some other structural famil-ies, that a very important difference between an endo-glucanase and a cellobiohydrolase is the size of such loops In a cellobiohydrolase, they are generally lon-ger, and form a tunnel that encloses the catalytic resi-dues Substrate usually reaches the active site by threading itself in from the end of the tunnel In con-trast, an endoglucanase has shorter loops that define a more open binding cleft, and allow more direct access
of an intact cellulose chain
Among the fungi that have a family 7 cellobiohydro-lase, it is the major enzyme in the cellulase mixture secreted The first member of the family for which the structure was determined was the cellobiohydrolase of Trichoderma reesei (a clonal derivative of Hypocrea jecorina), Tr_Cel7A, formerly called CBH 1 [9] Three acidic residues (Glu212, Asp214 and Glu217) were shown to be responsible for cleavage of the cellulose chain Further studies allowed a complete mapping of cellulose binding along the 50 A˚-long active site tunnel [10,11] Tr_Cel7A binds 10 glucosyl units in subsites )7 to +3 (numbering starts from the point of glycosi-dic bond cleavage, between )1 and +1; negative num-bers indicate the nonreducing end of the cellulose chain, and positive numbers, the reducing end [12]) The +1 and +2 sites are often designated as the
‘product sites’, as they bind the cellobiose unit that will
-2 -1 +1 +2
Fig 1 Binding of disaccharides to Pc_Cel7D Overall structure of Pc_Cel7D with cellobiose bound in the +1 ⁄ +2 sites Backbone of the enzyme’s catalytic domain is coloured terracotta, aromatic side chains that form cellulose-binding subsites are green, and the three acidic residues involved in catalysis are red Cellobiose is indicated by a ball-and-stick model coloured light blue Numbers indicate the position of some of the glucosyl-binding subsites.
Trang 3be cleaved off at the reducing end of the chain
(Fig 1) These sites are placed close to the exit of the
binding site cleft⁄ tunnel, which should simplify the
subsequent release of the disaccharide However,
prod-uct inhibition is commonly observed for
cellobiohydro-lases [13–16]
Structures are also known for three endoglucanases
of family 7, from T reesei [17], Humicola insolens [18]
and Fusarium oxysporum [19] The active sites of these
enzymes are very similar and the enzymes are believed
to use the same catalytic mechanism as the
cellobio-hydrolases As expected, the loops flanking the
cellu-lose-binding cleft in each case are significantly shorter,
leaving the active sites completely open to solvent
Cellobiohydrolase Cel7D (previously called CBH 58)
is the major cellobiohydrolase produced by the
basi-diomycete Phanerochaete chrysosporium under most
growth conditions [20] We recently solved the
struc-ture of Pc_Cel7D, and showed that it is similar to
Tr_Cel7A [21] The catalytic residues were identified as
Glu207, Asp209 and Glu212 Nearly all interacting
residues of Tr_Cel7A are conserved, which suggested
that Pc_Cel7D would bind cellulose in much the same
way However, several deletions make the binding
tunnel slightly more open in Pc_Cel7D
A recent comparative study revealed striking
differ-ences in the activity on insoluble model substrates:
although Pc_Cel7D had only slightly higher activity on
cellotetraose, it hydrolysed amorphous and bacterial
microcrystalline cellulose eight times and 4.4 times
fas-ter, respectively, than Tr_Cel7A Enzyme kinetics on
p-nitrophenyl lactoside gave similar kcat values for the
two enzymes; however, Pc_Cel7D showed a threefold
higher Km(and hence threefold lower kcat⁄ Km) as well
as reduced cellobiose inhibition (eight times higher Ki)
[22] Furthermore, estimation of specificity constants
(kcat⁄ Km) for dinitrophenyl-cellooligosaccharides with
2–5 glucose units, pointed at differences between the
enzymes in the relative contribution of intrinsic
bind-ing energy to catalysis at subsites )3 to )5 Another
study revealed differences in the binding specificity for
cellobiose and lactose, presumably at the product sites
+1⁄ +2 While Tr_Cel7A prefers binding of cellobiose
to lactose, the opposite is true for Pc_Cel7D [23]
As part of global efforts to replace fossil fuels with
renewable energy sources, cellulases have received
increasing attention as a possible means of converting
cellulosic biomass to fermentable sugars for ethanol
production [24] However, the enzyme cost is a critical
factor, and improvements in the efficiency of the
pro-cess will directly influence whether such ‘bioethanol’
can effectively compete with petroleum [25] The major
industrial source of cellulase enzymes at present is
T reesei [26] Deletion of individual cellulase genes in
T reesei showed that Tr_Cel7A was rate limiting in the degradation of crystalline cellulose in the fungal system [27] Understanding the molecular details of how the Cel7 enzymes work thus lies at the heart of finding the best solution in future applications
In the present paper, we report three structures of Pc_Cel7D in complex with disaccharides: the product (cellobiose) and two inhibitors (lactose and cellobio-imidazole) These structures provide a picture of two different glycosyl binding modes, as well as explaining the differences in affinity between the two natural sugars A structure obtained in the presence of cellobi-ose, methyl 4-S-b-cellobiosyl-4-thio-b-cellobioside (GG-S-GG), Tris⁄ HCl and calcium revealed that Tris binds
in the active site In kinetic studies, we show that Pc_Cel7D is in fact inhibited by both Tris and calcium
at the concentrations used in the crystallization; this
is the first report of such behaviour within the family
Results
Overall structures Deglycosylated Pc_Cel7D catalytic module was crystal-lized in the presence of two natural disaccharides, cell-obiose and lactose, as well as with cellobioimidazole, a compound that mimic the transition state of some cel-lulases [28] The crystals were isomorphous with pre-vious ones [21] and complete diffraction data sets to 1.7 A˚ resolution or better could be collected using syn-chrotron radiation In all three cases, clear electron density was found for the bound ligand prior to its inclusion in the models (Fig 2) Statistics relating to the diffraction data and the final refined models are summarized in Table 1 Each model contains the com-plete catalytic module of Pc_Cel7D (residues 1–431),
an N-acetylglucosamine residue bound to Asn286, one molecule of the respective ligand and a number of bound waters The protein structures are very similar
to each other and to the published structure of Pc_Cel7D {Protein Data Bank (PDB) [29] entry code 1GPI [21]} with overall r.m.s differences of 0.2–0.3 A˚ when all Ca atoms are compared pair-wise
Binding of cellobiose to Pc_Cel7D Product inhibition in Pc_Cel7D is consistent with the observed binding of cellobiose in the +1⁄ +2 (pro-duct) sites of Pc_Cel7D (Figs 1, 2A and 3A) The nonreducing end of the disaccharide is in the +1 site; this glucosyl unit shows the ‘classical’ stacking on a tryptophan residue that is a feature in many proteins
Trang 4interacting with carbohydrates [30] The hydrophobic
B-face of the sugar thus makes a number of nonpolar
contacts with the indole ring of Trp373 (Fig 3A) The
interactions on the opposite (A) face of this
carbohy-drate unit are more polar O2 interacts with the
main-chain carbonyl oxygen of Asp248 O3 and O4 are
linked to the catalytic acid Glu212 via hydrogen bonds
with water In addition, the guanidino group of
Arg240 forms hydrogen bonds with O5 and O6 The
electron density for the side-chain atoms of Arg240 is
slightly weaker than average Both the structural
set-ting and the density suggest that the interactions of
Arg240 with sugar compete with a salt link to Asp248,
and a hydrogen bond to Gln172 Fewer interactions
are seen in subsite +2, and electron density of this
glycosyl unit is also somewhat weaker than that
observed for the +1 sugar The observed interactions
are on the same side of the cleft as those in the +1
subsite The guanidino group of Arg391 is within
hydrogen bonding distance to O1, O5 and O6 of the
sugar O1 also interacts with Asp336, and O6 with a
solvent molecule There is, however, no aromatic
stacking in the +2 subsite
Both glucosyl rings adopt a regular 4C1 chair, i.e a
favourable conformation in solution The planes of
the two sugar units have opposite orientations, with
torsion angles (/¼)78, w ¼ +120) that deviate slightly from those observed in the small-molecule crystal structure of cellotetraose ()93, +96, and )93, +86) [31] Of the inter-residue interactions that stabil-ize cellulose chains, only the O3i+1–O5i hydrogen bond is present; that between O6i+1 and O2i is lack-ing The less common gauche-gauche conformation of the exocyclic C6–O6 bond is apparently stabilized by its interaction with Arg391, which is preferred to an intramolecular one with O2 The sugar is tightly sand-wiched between the walls of the product sites by the interactions with protein described above The hydro-xyls along one edge of the disaccharide point into the binding cleft, where several water molecules are found; hydroxyls along the other edge point out toward the bulk solvent
Binding of lactose to Pc_Cel7D Lactose is an effective competitive inhibitor of Pc_Cel7D (Table 2) The only chemical difference between this disaccharide and cellobiose is the confi-guration at C4 in the galactosyl unit: the hydroxyl group is equatorial in cellobiose, and axial in lactose
As might be expected, lactose binds in the +1⁄ +2 subsites in a manner very similar to that described
+2 +1
-1 -2
-3 -4
-5
D GG-S-GG + TRIS + cellobiose
Fig 2 Electron density for ligands bound in Pc_Cel7D Final 2F o -F c maps contoured at 1 r, are shown for (A) cellobiose (B) lactose (C) cello-bioimidazole and (D) GG-S-GG, Tris and cellobiose The numbers for the glucosyl-binding subsites indicate the location within the substrate binding tunnel of Pc_Cel7D.
Trang 5above for cellobiose (Figs 3B and 4A) However, the
axial placement of O4 allows the disaccharide to make
a direct hydrogen bond to Arg240 The guanidino
group of that side chain has rotated slightly, so that
it interacts with O4 and O5, instead of O5 and O6
(Fig 3B) At the same time, Arg240 can make more
favourable interactions with Gln172 and Asp248 O6
now appears to lack a fixed hydrogen-bonding
part-ner The interactions of the glucosyl unit in the +2
site are the same as those observed for cellobiose
However, the electron density for both sugar and
protein in the immediate area is significantly better
than that observed for cellobiose (Fig 2A and B),
and the temperature factors for the ligand are
corre-spondingly lower (Table 1) These differences provide
a structural basis for the observation that Pc_Cel7D
binds more tightly to lactose than to cellobiose
(Table 2)
Binding of cellobioimidazole in Pc_Cel7D
Monosaccharide-derived imidazoles such as
cellobio-imidazole (Fig 2C), feature an sp2-hybridized
ano-meric centre and a charge distribution that mimics the
transition state of some exoglycosidase reactions
[32,33] Cellobioimidazole is apparently not sufficiently
compatible with the transition state of Pc_Cel7D to
promote its binding in the catalytic substrate sites
)2 ⁄ )1 The preferred binding is instead in sites
+1⁄ +2, as was seen for the other two disaccharides
(Fig 3) The cellobioimidazole is, however, shifted
more than 2 A˚ along the cleft, toward the catalytic centre (Fig 4A)
The glucosyl unit in the +1 site continues to make good stacking interactions with Trp373, although it now lies more directly against the six-membered ring
of the indole O2 maintains the hydrogen bond to
248-O, but the sugar oxygen is displaced 1 A˚ from its position in the cellobiose and lactose complexes; this is the only one of the polar interactions that is preserved
in this site The hydrogen-bonding capacity of O3 is saturated by interactions with the side chains of His223, Asp209 and Glu212, and with a solvent mole-cule O4 also makes a hydrogen bond with Glu212, but O6 appears to have no hydrogen-bonding partner
in the enzyme Like the sugars of cellobiose and lac-tose, the glucosyl unit in cellobioimidazole adopts a regular4C1chair conformation
In the +2 site, the glucoimidazole ring makes hydrogen bonds to Arg240 and Arg391, as well as to several solvent molecules Under the crystallization conditions (pH 7.0), only a small fraction of the cello-bioimidazole will be protonated (pKa 6.1 [34]), and charge–charge repulsion is apparently not a problem Unlike the glucosyl and galactosyl units, the glucoimi-dazole moiety cannot adopt a 4C1 chair conformation because of its C1–N5 double bond The most favour-able solution conformation is the 4H3 half-chair observed in the crystal and NMR structures of glucoimidazole alone, although several other confor-mations are also possible within its pseudo-rotational sequence [35] In the complex with Pc_Cel7D, the
A
cellobiose
212
391
240
207 373
C
cellobioimidazole
His223 212
207 209
240 373
Arg391
Asp336
Asp248
Arg240 Gln172 Glu207
nucleophile
Asp209 Trp373 Arg256
Glu212
acid/base
B
lactose
+2
+1
Fig 3 Interactions between the disaccharides and Pc_Cel7D Hydrogen-bonding interactions are shown for (A) cellobiose (B) lactose and (C) cellobioimidazole In each case, the protein is shown with gold carbon atoms, while those of the ligand are yellow Hydrogen bonds are indi-cated by cyan-coloured ‘bubbled’ lines Water molecules interacting with protein and ligand are small light-blue spheres.
Trang 6conformation is closest to an envelope form, with C3
out of plane
The electron density (Fig 2C) and temperature
fac-tors (Table 1) for cellobioimidazole, as well as its good
interactions with protein, suggest that it should be an
effective cellobiohydrolase inhibitor No affinity
meas-urements are yet available for this compound with
Pc_Cel7D, but the related Tr_Cel7A is indeed inhibited
by cellobioimidazole, although, at least at pH 5.7,
bind-ing is weaker than for cellobiose (Table 2) [36] Due to
the close proximity of the imidazole ring to the
guanidi-no groups of arginines 256 and 391, it seems likely that
the ligand will be a better inhibitor at pH 7 or higher,
when the glucoimidazole ring is unprotonated, than at lower pH More biochemical data are obviously needed
Binding of thio-linked substrate analogue GG-S-GG and cellobiose
Crystals of Pc_Cel7D were soaked with a combination
of cellobiose and the thio-linked sugar GG-S-GG, in hopes of obtaining a complex that included sugars bound in both ends of the active-site cleft As seen from the electron density in Fig 2D, the product sites +1⁄ +2 were again completely occupied with disac-charide, in the position observed with cellobiose alone
Table 2 Selected binding and kinetic constants for Pc_Cel7D and Tr_Cel7A.
Enzyme
Kd, cellobiose (l M )
Kd, lactose (l M )
Ki, cellobio-imidazole (l M )
Km, pNP-Lac (l M )
kcat, pNP-Lac (s)1)
a Kdvalues at pH 5.0 and 25 C from displacement chromatography experiments with Pc_Cel7D immobilized on silica, as published by Hen-riksson et al [23] b Michaelis–Menten kinetic parameters at pH 5.0 and 25 C [23] c Kdat pH 5.0 and 25 C determined by protein differ-ence spectroscopy [16]. dNon-competitive K i at pH 5.7 and 30 C from inhibition experiments using 2-chloro-nitrophenyl b-lactoside as substrate [36] NA, data not available.
Table 1 Data collection and refinement statistics The space group was C2 Statistics for the highest resolution shell are given in paren-theses A stringent boundary Ramachandran plot was used [47] Data collection statistics were taken from TRUNCATE [48] Other values for the refined structures were calculated using MOLEMAN 2 [49].
Data collection
Complex
Cellobiose, Tris, GG-S-GG
Resolution (A ˚ ) 50–1.70 (1.73–1.70) 50–1.60 (1.63–1.60) 43–1.70 (1.79–1.7) 96.321–1.7 (1.79–1.70)
Refinement
(completeness,%)
Number of protein atoms (Average B, A˚2) 3198 (24.7) 3198 (22.2) 3198 (17.5) 3198 (20.6)
Number of water molecules (Average B, A ˚ 2 ) 106 (27.9) 278 (31.2) 180 (21.9) 389 (30.8)
(Average B, A˚2)e
Number of ligand atoms (Average B, A ˚ 2 ) 23 (33.7) 23 (24.0) 25 (16.9) 31 (24.2)
Trang 7The other side of the cleft contains additional electron
density not observed in the other Pc_Cel7D structures,
which may indicate binding of the longer sugar at low
occupancy Although the shape of the density in sites
)4 and )5 bears strong resemblance to glucose rings,
on the whole it is too weak to allow unambiguous
modelling of this ligand within the active site The low
occupancy is almost certainly due to the limited crystal
soaking time used (2 min); the crystals deteriorated at
longer soaking times Co-crystallization could not be
used because the long exposure time would lead to
hydrolysis of the O-glycosidic bonds in the ligand We
are currently seeking other solutions to this problem
However, clear electron density in the immediate vicinity of the catalytic residues of this structure was only compatible with Tris, among the known crystal-lization reagents Inhibition by Tris had not been reported previously for Cel7 enzymes
Inhibition experiments The discovery of Tris density in the active site promp-ted us to undertake a systematic study of the com-ponents of the crystallization solution Inhibition experiments at pH 7.0 using p-nitrophenyl lactoside as substrate (summarized in Fig 5) showed that both
10 mm Tris⁄ HCl and 5 mm CaCl2 individually inhibit Pc_Cel7D As there was no inhibition with 10 mm NaCl, the Tris and the calcium ions are the inhibiting species
Comparison with ligand binding in Tr_Cel7A
To date, five structures have been published for the related cellobiohydrolase of T reesei (Tr_Cel7A) with carbohydrates bound in sites +1⁄ +2: the wild-type enzyme with the inhibitor o-iodo-benzyl-b-d-thio-glucoside (IBTG; PDB entry 1CEL [9]), an inactive
B
A
C
Fig 4 Superposition of sugar residues bound in the +1 ⁄ +2 sites of Pc_Cel7D and Tr_Cel7A Selected residues of Pc_Cel7D are shown with gold carbon atoms and of Tr_Cel7A with blue carbon atoms (A) Superposition of the three disaccharides as bound by Pc_Cel7D Cellobiose (magenta), lactose (lilac) and cellobioimidazole (cyan) are shown as ball-and-stick representations Only cellobioimidazole enters directly into the active site, forming hydrogen bonds to the catalytic acid Glu212 The interactions of O2 with 248-O in the +1 site, and Arg391 with O6 in the +2 site, are the only direct polar interactions found in all three complexes (Results) Two additional conserved interactions are shown, in which water molecules medi-ate links between sugar hydroxyls and protein residues at the deep-est point of the binding cleft The O6 hydroxyl in subsite +2 thus also interacts with a water molecule bound to Asp251 OD2 A sec-ond water molecule links Thr221-OG1 to O2 in site +1 These inter-actions hold O2 of the +1 subsite and O6 of the +2 subsite in very similar positions in all three complexes (B) Cellobiose binding near the catalytic residues in Pc_Cel7D (magenta ligand) is shown together with the complex of cellobiose with Tr_Cel7A (yellow lig-and) In this binding mode there is room for water (pale green spheres) between the O4 hydroxyl of the hexose in site +1 and the catalytic acid ⁄ base (Glu212 in Pc_Cel7D) (C) Superposition of avail-able cellobiohydrolase structures with sugars bound in the +1 ⁄ +2 sites highlighting the existence of two discrete binding modes The bound sugar residues are colour-ramped using a rainbow, with blue indicating the position closest to the point of cleavage, and red, that farthest away The identity of each complex is indicated by coloured boxes Conserved interactions involving water are also shown.
Trang 8E212Q mutant with cellobiose (3CEL [10]),
cellotetra-ose (5CEL [11]) and cellopentacellotetra-ose (6CEL [11]), and a
second inactive mutant (E217Q) with cellobiose
(together with cellohexaose bound in sites )7 to )2;
7CEL [11]) The catalytic domains of Tr_Cel7A and
Pc_Cel7D share 55% amino-acid sequence identity,
which provides a good basis for detailed comparison
of the two enzymes
The complex of Tr_Cel7A with cellobiose (3CEL)
can be superimposed on that of Pc_Cel7D using a
cut-off of 0.7 A˚, giving an r.m.s difference of 0.4 A˚
for 267 matching Ca atoms ( 60% of the total)
The most similar portions of the enzymes represent
the core b-sheet structure as well as the highly
con-served residues of the active site Cellobiose is bound
in an equivalent position in the two enzymes
(< 0.8 A˚ difference for all atoms); the position of
O6 within the +2 site is identical (Fig 4B)
Although this Tr_Cel7A structure actually represents
an inactive mutant, the mutated residue (equivalent
to Pc_Cel7D’s Glu207) is not directly involved in lig-and binding, lig-and does not appear to complicate the comparison The most significant differences between the two complexes result from a deletion in one of the active-site loops of Pc_Cel7D (Fig 6) The role
of Arg240 in binding to O5 and O6 in the +1 site
is thus assumed by Arg251 in Tr_Cel7A The main-chain atoms of Arg251 are 3 A˚ away from those
of Arg240, but the functional guanidino groups are similarly placed and serve a similar purpose in the two enzymes However, Arg251 in Tr_Cel7A is sup-ported by a better local network of hydrogen bonds, including Thr246 and Asp259 The longer loop in Tr_Cel7A also provides one additional direct hydro-gen bond to the ligand: Thr246 interacts with O6 in the +1 site On the other hand, a deletion in Tr_Cel7A results in the loss of an interaction at O1
in site +2, which can be provided by Asp336 in Pc_Cel7D (Fig 6) All of the other interactions that Pc_Cel7D makes with cellobiose are found intact in the complex with Tr_Cel7A Better hydrogen bond-ing, together with the more enclosed Tr_Cel7A active site, provides a reasonable explanation for why cello-biose binds more tightly to this enzyme than to Pc_Cel7D (Table 2), and so there is less product inhibition in the latter enzyme
Fig 6 Comparison of the Pc_Cel7D and Tr_Cel7A structures near the exit of the cellulose-binding tunnel The complex of Pc_Cel7D with cellobiose is in gold and coral carbons, while Tr_Cel7A is in green The sugar is embraced in Tr_Cel7A by the 245–250 loop (seen at the upper left), but not in Pc_Cel7D that has a six-residue deletion here Pro258 in Tr_Cel7A may hold Arg251 out of reach for O4 of lactose At the bottom of the figure it is seen that the inser-tion of Asp336 in Pc_Cel7D results in differences in main-chain con-formation and provides an additional hydrogen bond with the reducing-end hydroxyl of a bound cellulose chain.
B
A
Fig 5 Inhibition of Pc_Cel7D by Tris and other compounds.
Absorbance at 400 nm was measured after a 30-min incubation of
Pc_Cel7D with pNP-Lac at 30 C, pH 7.0, as described in
Experi-mental procedures (A) h, No inhibitor; ·, 10 m M NaCl; +, 10 m M
Tris ⁄ HCl; –, 5 m M CaCl2; *, 5 m M CaCl2and 10 m M Tris ⁄ HCl (B)
h , No inhibitor; n, 0.1 m M cellobiose; s, 0.1 m M cellobiose and
10 m M Tris ⁄ HCl; e, 0.1 m M cellobiose, 10 m M Tris ⁄ HCl and 5 m M
CaCl2.
Trang 9There is presently no structure available for
Tr_Cel7A in complex with lactose, but some points
seem clear in a comparison of the two enzymes In
Pc_Ce7D, the flexibility of the Arg240 side chain is an
important factor in binding of the various
disaccha-rides In the lactose complex particularly, Arg240 has
adapted its conformation to give a good
hydrogen-bonding network that includes the axial O4 of the
galactosyl unit Possibilities are different for the
Arg251 in Tr_Cel7A, since both steric and
hydrogen-bonding options will be altered locally In Pc_Cel7D,
lactose also has an additional H-bond from O1 to
Asp336 in the +2 site Apparently, the combined
differences do not favour the binding of lactose over
cellobiose by Tr_Cel7A (Table 2)
Discussion
Our structural results provide a good framework for
understanding why Pc_Cel7D binds lactose more
tightly than cellobiose, and why Tr_Cel7A exhibits the
opposite behaviour Furthermore, the distinct mode of
binding exhibited by the complex of Pc_Cel7D with
cellobioimidazole prompted a closer look at the
avail-able structural data on binding in the +1 and +2 sites
in these enzymes (Fig 4C) The nonreducing end of
the disaccharide in each of the available complex
struc-tures is in the +1 site, and the reducing end in the +2
site However, two quite distinct binding modes are
clearly present In one scenario, the hexose in site +1
is close to the active centre, with its O4 hydroxyl
bound to the catalytic acid Glu212 (equivalent to 217
in Tr_Cel7A) This type of binding is found in the
Pc_Cel7D⁄ imidazole complex, and in the complexes of
Tr_Cel7A with IBTG, cellopentaose, and cellobiose +
cellohexaose (1CEL, 6CEL, 7CEL) In the second
mode, the sugar is shifted 2 A˚ away from the
cata-lytic centre, leaving room for a water molecule between
the sugar and the catalytic acid This type of binding
is observed for cellobiose and lactose in Pc_Cel7D,
and for complexes of cellobiose or cellotetraose with
Tr_Cel7A (3CEL and 5CEL, respectively) The
loca-tion of O2 in the +1 site, and of O6 in the +2 site, is
very similar in all structures; the two primary binding
modes appear to result from a pivoting motion around
these points As the disaccharide moves away from the
catalytic residues, the sugar in the +1 site moves out
toward the bulk solvent, and the sugar in the +2 site
moves deeper into the cleft⁄ tunnel of the enzymes
Processive hydrolysis of cellulose requires that the
enzyme can slide along a cellulose chain The tunnels
of Pc_Cel7D and Tr_Cel7A are wide enough to allow
the passage of the chain, apparently without need for
conformational changes in the protein [11,21] When the reducing end of the chain has passed the active centre and entered into the product binding sites, the glucose residue at site )1 still has sufficient space to remain in the most stable 4C1 chair conformation However, in order for hydrolysis to take place the )1 glucosyl must approach the catalytic nucleophile at the bottom of the catalytic centre; this requires a flip from the chair into the boat conformation, and a concomit-ant bending of the cellulose chain at this position Our observations hint at events near the active site during catalysis We propose that the docking mode where the disaccharide unit is placed immediately at the active site (as observed, e.g for the complex of Pc_Cel7D with cellobioimidazole) represents a ‘cut’ mode We will refer to the other docking position, that where the disaccharide is slightly further away (as for the cellobiose and lactose complexes), as the ‘slide’ mode
For the cellulose chain to be cleaved, it must first dock in the ‘cut’ mode, and the )1 glucosyl must flip from a chair to a boat conformation The O3 hydroxyl
in site +1 then points down towards the deepest part
of the cleft, and interacts directly with the acid⁄ base Glu212-OE1 It is now also within hydrogen-bonding distance of the catalytically important Asp209 and His223 The other carboxylate oxygen of Glu212 hydrogen-bonds to O4 In a true enzyme–substrate complex, this O4 hydroxyl would actually be the gly-cosidic oxygen that links the sugar in site +1 to that
in site )1; the hydrogen bond between Glu212 and O4
is suggestive of the protonation of the glycosidic oxy-gen in the transition state In the transition state, the reducing end of the cellulose substrate (i.e the cello-biosyl unit in sites +1⁄ +2) remains bound in the ‘cut’ mode, with the glycosidic oxygen bound to Glu212 Once the cellulose chain is cleaved, the cellobiose product can remain bound, but pivots into the ‘slide’ mode Now, the positions previously occupied by the O3 and O4 hydroxyls are filled by water molecules to which O3 and O4 bind The water molecule that lies between O4 of the hexose in site +1 and the acid⁄ base Glu212 is compatible with the existence of the inter-mediate, and well positioned to perform a nucleophilic attack on its anomeric carbon Therefore, it seems possible that cleavage of the intermediate is followed
by release of the product, rather than the product necessarily leaving prior to hydrolysis, as has always been proposed Indeed, the cellobiose could be essential
to proper positioning of the catalytic water After the water attack, the glucosyl unit at the new reducing end
of the cellulose chain (that in the )1 site) will have a new hydroxyl group in the b-configuration When the
Trang 10sugar unit flips back from the boat conformation to an
ordinary 4C1 chair, this would be expected to create
steric problems that would force the cellobiose product
(rather than the longer and more firmly bound cellulose
chain on the substrate side) to leave the site Thus the
energy released when the high-energy glycosyl-diester
bond is cleaved would provide kinetic energy used to
drive product release Such a mechanism might explain
why cellobiose is a much weaker inhibitor of Tr_Cel7A
( 80-fold difference in Ki) when it acts processively
on cellulose than with soluble substrates [37]
One might expect that the enzyme had evolved for
optimal interactions with the cellobiosyl unit in the
‘cut’ mode, in order to maximize transition-state
stabil-ization Since cellobiose clearly makes more favourable
interactions with Pc_Cel7D in the ‘slide’ mode,
stabil-izing the transition state cannot be the sole
considera-tion for this enzyme A key residue in stabilizing the
‘slide’ mode in Pc_Cel7D, Asp336, is located at the
very end of the binding cleft⁄ tunnel (Fig 6) The
cor-responding residue is deleted in Tr_Cel7A An
align-ment of 53 nonredundant sequences retrieved from the
ProDom server (http://prodes.toulouse.inra.fr/prodom/
2004.1/html/home.php) indicates that family 7
endo-glucanases lack this structural motif, although it is
rather well conserved in the cellobiohydrolases In 31
out of 41 cellobiohydrolase sequences, the segment has
the same length, and the aspartate is conserved In
another five the length is conserved, but not the
aspar-tate In two of these, the aspartate is replaced with
glu-tamate that might play a similar role Four sequences
are shorter (by one residue), including three enzymes
of Trichoderma species (T reesei, T viride, T
harzia-num) and one from Thermoascus aurantiacus
(Swiss-Prot accession code Q96UR5) There is also a single
Cel7 sequence with a one-residue insertion; this
seg-ment includes two glutamates, but no aspartate
(Lep-tosphaeria maculans Cel2, Q9P8K7) We anticipate
that the differences will be indicative of different
kin-etic properties in the respective enzymes Among the
enzymes that can stabilize the ‘slide’ mode in this way,
one might also expect a reduction of transglycosylation
activity, since the product would be too distant to
per-form the reverse reaction
The Pc_Cel7D structure was used previously for
homology modelling of the other five family 7
iso-enzymes in P chrysosporium [21] We predicted that
the catalytic properties of Pc_Cel7C, E and F would
be very similar to those of Pc_Cel7D ( 80% identity),
while Pc_Cel7A and B were expected to be more
distinct (66% identity) Re-evaluation of the models
with the present structural data indicates that the two
residues (Arg240 and D336) implicated as important
for binding in the product sites are conserved in C, E and F, but not in A and B isozymes Arg240 is replaced by Ser (in A) or Ala (in B), while Asp336 is replaced by Glu (in A) or Gly (in B) The differences would be expected to reflect different kinetic proper-ties, and possible endoglucanase activity
Our data also provide the new information that Pc_Cel7D is inhibited by both Tris and calcium As these conditions resemble those in the crystallization solutions used here, it is not surprising that Tris is observed in the active site of the complex with
GG-S-GG, although the tetrasaccharide is observed at only low occupancy (Fig 2D) The position of Tris near the nucleophile Glu207 and its partner Asp209, as well as the acid⁄ base Glu212, provides a clear explanation for the inhibition Re-inspection of previous data con-firmed that Tris is not present in the structures with apo enzyme or the disaccharides alone, indicating that some degree of synergy exists in its binding with the thio-linked sugar Comparison of the catalytic-site regions suggests that these compounds will also bind
to and inhibit Tr_Cel7A Such inhibition has not pre-viously been reported for enzymes in glycoside hydro-lase family 7, although unrelated proteins with similar catalytic sites, such as the family 13 amylase [38] are known to be inhibited by Tris Although the amylase has a completely different structure, based on a (b⁄ a)8 barrel, it too is a retaining enzyme that binds Tris in the)1 site in the immediate vicinity of the nucleophile and catalytic acid
Experimental procedures
Preparation of protein, crystallization and data collection
Intact Cel7D protein from P chrysosporium was the kind gift of Gunnar Johansson, Department Biochemistry, Uppsala University Preparation of the deglycosylated cata-lytic module of Cel7D has been described previously [21] Hanging-drop vapour diffusion experiments included
18 mgÆmL)1protein, 10 mm Tris⁄ HCl pH 7.0, 5 mm CaCl2, 15–22.5% polyethylene glycol 5000 and 12% glycerol Sin-gle-soaking experiments of Cel7D crystals were performed with 10 mm cellobiose, 10 mm lactose (Sigma, St Louis,
MO, USA) and 5 mm cellobioimidazole {(5R,6R,7S,8S)-6-(b-d-glucopyranosyloxy)-5,6,7,8-tetrahydro-5-[(hydroxy)methyl] imidazo[1,2-a] pyridine-7,8-diol [36]}, respectively A dou-ble-soak experiment was performed with 10 mm cellobiose followed by 0.5 mm of the thio-linked cellotetraoside, GG-S-GG Soaking time of the shorter ligands was almost
10 min, but even after 1 day these crystals were stable However, in the case of the GG-S-GG soaks, crystals were