Báo cáo khoa học: Investigation of the substrate specificity of a b-glycosidase from Spodoptera frugiperda using site-directed mutagenesis and bioenergetics analysis pdf

Terra Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil The specificity of the Spodoptera frugiperda digestive b-glycosidase Sfbgly50 for

Investigation of the substrate specificity of a b-glycosidase from

bioenergetics analysis

Sandro R Marana, Eduardo H P Andrade, Cle´lia Ferreira and Walter R Terra

Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil

The specificity of the Spodoptera frugiperda digestive

b-glycosidase (Sfbgly50) for fucosides, glucosides and

galactosides is determined by noncovalent interactions of

glycone 6-OH and glycone 4-OH with the active-site residues

Q39 and E451 Site-directed mutagenesis and enzyme

steady-state kinetics were described, showing that replacement of

E451 with glutamine increased the preference of Sfbgly50

for glucosides in comparison to galactosides, whereas

replacing E451 with serine had the opposite effect In

contrast, the replacement of E451 with aspartate did not

change Sfbgly50 specificity The energy of the interactions

formed by these different residues with the axial and

equa-torial glycone 4-OH were also measured, showing that the

increase in preference for galactosides resulted from a larger

energy decrease in the interaction with equatorial 4-OH

than with axial 4-OH (22.6 vs 13.9 kJÆmol)1), whereas the

increase in preference for glucosides was caused by an

energy reduction in the interaction with the axial 4-OH (5.1 kJÆmol)1) The introduction of glutamine at position

451 or of asparagine at position 39 increased the preference

of Sfbgly50 for fucosides in comparison to galactosides, whereas the presence of aspartate or serine at position 451 had less effect on this preference The hydrolysis of fucosides was favored because glutamine at position 451 increased a steric hindrance with 6-OH of 7.1 kJÆmol)1and asparagine

at position 39 disrupted a favorable interaction with this same hydroxyl In conclusion, it is proposed that the spe-cificity of new b-glycosidase mutants can be predicted by combining and adding energy of the enzyme–substrate interactions evaluated in the present study

Keywords: b-glycosidase; bioenergetics analysis; enzyme specificity; glycoside hydrolase; site-directed mutagenesis

b-glycosidases from glycoside hydrolase family 1 are

enzymes that remove monosaccharides from the

nonreduc-ing end of di- and/or oligosaccharides The nonreducnonreduc-ing

monosaccharide residue binds at the glycone subsite (subsite

)1), whereas the remaining part of the substrate is

accommodated by the aglycone subsite

be composed of several subsites (+1, +2, +3, etc.)

According to the CAZy database, family 1 currently

comprises 427 b-glycosidases, with 3D structural data being

available for 12 [1] All family 1 b-glycosidases share the

same tertiary structure [(b/a)8 barrel]; they are

configur-ation-retaining glycosidases, the catalytic activity of which

depends on two glutamic acid residues, one positioned after the b strand 4 (catalytic proton donor) and the other after the b strand 7 (catalytic nucleophile) Family 1 comprises enzymes with 14 different EC numbers

hydro-lysis of substrates presenting a variety of glycones (mono-saccharides such as glucose, galactose, fucose, mannose, 6-phosphoglucose and 6-phosphogalactose) and aglycones (monosaccharides, oligosaccharides, alkyl and aryl moiet-ies) [1] This broad substrate specificity makes family 1 an interesting model for using to study the molecular basis of the enzymatic specificity

Having a better understanding of the molecular basis of b-glycosidase specificity would result in an improved knowledge of the physiological role of these enzymes, as well as contributing to the design of b-glycosidases with novel specificities

Enzymatic specificity mostly relies on noncovalent inter-actions between amino acid residues within enzyme active sites and groups of the reactant transition state (S
) [2]; the strength of the noncovalent interaction between enzyme active sites and S
determines the stability of the enzyme– transition state (ES
) complex and the rate of the reaction Thus, in theory, modifications of the enzymatic specificity could be accomplished by changing active-site residues The role of the noncovalent interactions between amino acid residues of the active site and glycone hydroxyls have already been studied in family 1 b-glycosidases from

Correspondence to S R Marana, Departamento de Bioquı´mica,

Instituto de Quı´mica, Universidade de Sa˜o Paulo, CP 26077,

Sa˜o Paulo, 05513-970, Brazil Fax: +55 11 38182186,

1Tel.: +55 11 30913810, E-mail: srmarana@iq.usp.br

Abbreviations: ES, enzyme–transition state complex; DG, activation

energy of the glycosylation step; DDG, differences in the activation

energy of the glycosylation steps; 3-OH, glycone hydroxyl 3; 4-OH,

glycone hydroxyl 4; 6-OH, glycone hydroxyl 6; NP, p-nitrophenyl;

S, reactant transition state.

Enzymes: digestive b-glycosidase (b- D -glucoside glucohydrolase) from

Spodoptera frugiperda (EC 3.2.1.21) GenBank access no.: AF052729.

(Received 28 March 2004, revised 24 August 2004,

accepted 3 September 2004)

Agrobacterium sp [3], in Pyrococcus furiosus [4], in lamb

(lactase-phlorizin hydrolase) [5], in guinea pig (citosolic

b-glucosidase) [6] and in Spodoptera frugiperda [7]

Nevertheless, how the substitution of active-site amino

acids would affect the energy of noncovalent interactions

with the substrate in the ES
complex is not easily

predictable Consequently, there are only a few published

studies that report changing the specificity of family 1

b-glycosidases by mutagenesis [8–12] In these studies,

residues interacting with the substrate were replaced with

a single amino acid The criterion applied when choosing the

amino acid residue to be introduced in the mutant enzyme

was based either on a sequence comparison with other

b-glycosidases presenting the desired specificity [10,11], or

on the hydrogen-bonding properties of the mutant residue

[12] Although these studies were successful in changing

substrate specificity, they do not provide us with a

significant basis for further experiments on changing the

specificity of family 1 b-glycosidases by mutagenesis, as only

one type of mutation was evaluated in those and,

further-more, its possible effects on the energy levels of noncovalent

interactions with the substrate were not included

Noncovalent interactions in the ES
complex between

two amino acid residues (E451 and Q39) and the glycone

hydroxyls 4 (4-OH) and 6 (6-OH) determine the preference

of the digestive b-glycosidase from S frugiperda (Sfbgly50)

for three substrates: fucosides, glucosides and galactosides

[7] It was shown that E451 forms stronger hydrogen bonds

with equatorial and axial 4-OH than Q39 E451 presents a

steric hindrance, whereas Q39 has a weak interaction with

6-OH [7]

The purpose of the present study was to substitute

residues Q39 and E451 of Sfbgly50, through site-directed

mutagenesis, for residues presenting hydrogen

bond-form-ing side-chains Steady-state kinetic data for the hydrolysis

of different substrates by Sfbgly50 mutants were then used

to calculate the energy of noncovalent interactions in the

ES
complex between different residues at positions 39 or

451 and glycone 4-OH or 6-OH The resulting data were

used to clarify the specificity of family 1 b-glycosidases, as

well as to predict the specificity of new Sfbgly50 mutants

Materials and methods

All reagents, unless otherwise specified, were purchased

from Sigma or Merck

Site-directed mutagenesis

Site-directed mutagenesis was performed using a plasmid

pT7-7 [13] containing an inserted DNA fragment coding

for the mature Sfbgly50 (pT7b50) [14] Experiments were

carried out according to the instructions included with the

QuikChange site-directed mutagenesis kit (Stratagene)

The sequence of the mutagenic primers contained a

common segment (underlined) and mutated codon (NNN,

in bold) Thus, the primer sequence used on mutations at

position 39 was 5¢-CGCTACAGCCTCCTACNNNAT

CGAAGGTGCTTGG-3¢, with AAC and GAG as the

mutated codons for Q39N and Q39E, respectively For

mutation at position E451, the primer sequence was

5¢-GGAGTCTAATGGACAACTTTNNNTGGATGGA

GGGTTATATTGAGCG-3¢, with GAC, CAA and TCA

as mutated codons for E451D, E451Q and E451S, respectively DNA sequencing was used to confirm the incorporation of the mutated codon in the pT7b50 Expression of the mutant Sfbgly50

The Sfbgly50 mutants were expressed in Novablue DE3 cells (Novagen), as described previously [7]

Purification of the recombinant Sfbgly50 The mutant Sfbgly50 was purified by hydrophobic chro-matography [7] followed by a second step of ion-exchange chromatography introduced as an additional polishing step The fractions containing b-glycosidase activity eluted in the first chromatography were pooled, dialyzed in 20 mM

triethanolamine buffer, pH 8.0, for 16 h at 4C and then loaded onto a ResourceQ column (Amersham Bioscience) The nonretained proteins were washed out in the dialysis buffer, while the retained proteins were eluted from the column using an NaCl gradient prepared in the same buffer The presence of the recombinant Sfbgly50 was detected using NPbglc [15] and its purity ascertained by SDS/PAGE followed by silver staining [16,17]

Protein determination was performed spectrophoto-metrically (absorbance at 280 nm) using e280¼

117 200M )1Æcm)1[18]

Kinetic analysis All assays were performed at 30C in 50 mM citrate-phosphate buffer, pH 6.0, and initial rate data were measured The hydrolysis of NPbglycosides was followed

by the release of p-nitrophenyl (NP) [15] The kinetic parameters (kcatand Km) were determined by employing 10 different substrate concentrations, and the data were fitted

to a Michaelis–Menten equation by using theENZFITTER

software (Elsevier-Biosoft, Cambridge, UK)

Calculation of the energy of noncovalent interactions

in the ES
complex Different Sfbgly50 mutants (Q39E, Q39N, E451Q, E451D and E451S) were prepared Noncovalent interactions involving any residue at position 39 were designated by /, while noncovalent interactions involving any residue at position 451 were designated by g Glycone hydroxyls 4 and

6 were designated 4 and 6, where e stands for an equatorial hydroxyl and a for an axial one Therefore, the interaction between any residue at position 39 and an equatorial 4-OH was called /4e, and between any residue at position 451 and

an equatorial 4-OH was called g4e Interactions involving these same residues and an axial 4-OH were named /4a and g4a, respectively Likewise, interactions between any residue

at position 39 and 6-OH were called /6 and between any residue at position 451 and 6-OH was designated g6 (Fig 1) X4a and X6 correspond to noncovalent inter-actions between any amino acid residue other than 39 and

451 with axial 4-OH and 6-OH, respectively

Interaction energy may be measured by using the method described previously [7] This method assumes that

noncovalent interactions formed by residues 39 and 451 are

independent and also compares the energy of ES
complexes

containing different sets of noncovalent interactions

be-tween E and S
The steps used to calculate the energy of

individual noncovalent interactions are described below

The reaction mechanism of family 1 b-glycosidases has a

glycosylation (from E + S to E–G) and a deglycosylation

(from E–G to E + G) step, each with an ES
complex, as

described by the following reaction [19]:

Eþ S ƒƒƒƒƒƒƒƒ!k1

k
1 ES
!k2

E-Gþ Ag !k3

Eþ G where S is formed by a glycone (G) covalently bound to

an aglycone (Ag), ES represents the Michaelis complex

and E–G the covalent intermediary (glycosyl-enzyme)

The following kinetic parameters are valid for that

reaction:

kcat¼ k2k3

k2þ k3

Km¼ðk
1þ k2Þk3

k1ðk2þ k3Þ

kcat=Km¼ k1k2

k2þ k
1 where kcat/Kmis the rate constant for the glycosylation step

and can be used to calculate its activation energy (DG
) DG

represents the difference of energy between the glycosylation

ES
complex and the E + S ground state Thus, as different

substrates (NPbglycosides) and enzymes (mutant or

wild-type) should present similar ground state energy, DDG
for

the glycosylation step represents the difference of the energy

between two ES
complexes Moreover, as the energy of an

ES
complex depends on the noncovalent interactions

between E and S
, DDG
values represent the sum of all noncovalent interactions that differ between the two ES
complexes

Therefore, differences in the activation energy of the glycosylation steps (DDG
), corresponding to a pair of different substrates hydrolyzed by the same enzyme, or to

a pair of different enzymes hydrolyzing the same substrate (Figs 2 and 3), were calculated by the following equation [2]:

DDGz¼ RT lnðc1=c2Þ where c1 and c2 are kcat/Km ratios determined from the hydrolysis of two different substrates by the same enzyme or from the hydrolysis of the same substrate by two different enzymes (R¼ 8.3144 JÆK)1Æmol)1and T¼ 303 K)

As detailed in Figs 2 and 3, all DDG
resulting from those comparisons are a sum of DG
Each DG
value represents the disruption of a noncovalent interaction owing to the substitution of residues 39 or 451 for an alanine residue or because of the lack of a glycone hydroxyl Thus, those DG
values actually represent the energy of noncovalent inter-actions in the ES
complex

The energy of noncovalent interactions /4e, /4a, /6, g4e, g4a and g6 can be calculated by isolating each

DG
through the subtraction of different DDG
values, as shown in Table 1 For example, the interaction g6 (inter-action between residue 451 and 6-OH) can be calculated

by subtracting DDGz

2 from DDGz

7, as detailed below: DDGz7
DDGz2¼ DGzg6þ DGz/6þ DGzX6
ðDGz/6þ DGzX6Þ

DDGz7
DDGz2¼ DGzg6

It should be noted that, by using this method, it was not possible to isolate the DG
corresponding to the energy of the hydrogen bond between residue 39 and 3-OH (/3), which was added to the energy of /4e and /4a interactions

Results and Discussion The induction of recombinant enzymes (mutants Q39E, Q39N, E451Q, E451D, E451S) was confirmed by SDS/ PAGE Then, the soluble material of the induced bacteria was used in the purification of the mutant enzymes through hydrophobic and ion-exchange chromatography Isolation

of the mutant enzymes was then confirmed by SDS/PAGE This procedure yielded  0.5 mg of mutants E451S and E451Q, 0.3 mg of mutant Q39E and 0.15 mg of mutants Q39N and E451D, from 0.5 L of bacterial culture Steady-state kinetic parameters for different Sfbgly50 mutants hydrolyzing several substrates were determined (Table 2) The replacement of residues E451 or Q39 with other residues mostly affects kcat, irrespective of the substrate used, suggesting that the measured energy differences mainly reflect differences in the ES
complex This finding corroborates the hypothesis that noncovalent interactions formed by glycone hydroxyls with the b-glycosidase active sites are stronger in ES
than in the ES complex [3]

All mutants have lower kcatand kcat/Kmvalues than wild-type Sfbgly50 (Table 2) Moreover, all mutants at position

Fig 1 Schematic diagram showing the noncovalent interactions (dotted

lines) involved in the substrate glycone (glc) binding with the wild-type

Spodoptera frugiperda digestive b-glycosidase (Sfbgly50) active site.

The interactions are identified as g6, g4e, /4e, /6 and /3 Residue

451 is represented by g, and / represents residue 39, e denotes an

equatorial hydroxyl, and 6, 4 and 3 indicate the glycone 6-OH, 4-OH

and 3-OH, respectively Interactions with the axial 4-OH (g4a, /4a)

were not represented Letters inside the boxes indicate residues

introduced through site-directed mutagenesis at positions 39 and 451.

The diagram was based on the structure of the Sinapis alba myrosinase

[24].

451 present higher kcat and kcat/Km values than mutant

E451A The same is true between mutants at position 39

and mutant Q39A As the rate constants are inversely

proportional to the ES
energy level [2], the results indicate

that, although the different residues introduced at positions

451 and 39 of Sfbgly50 are less effective on the stabilization

of the ES
complex than wild-type residues, they still retain

the ability to interact with S

The kcat/Kmratios were used to calculate DDG
within pairs of different substrates hydrolyzed by the same enzyme,

or pairs of different enzymes hydrolyzing the same substrate (Tables 3 and 4) These DDG
values are a sum of all noncovalent interactions that differ between two ES

complexes of the glycosylation step, each interaction corresponding to a DG
(Figs 2 and 3) The energy of all these noncovalent interactions between glycone hydroxyls

Fig 2 Free energy changes between enzyme–transition state (ES
) complexes formed by Spodoptera frugiperda digestive b-glycosidase (Sfbgly50) mutants at residue 39 ES
complexes, represented in the same column, were formed with the same substrate (glc, gal and fuc), whereas ES
complexes represented in the same row were formed with the same enzyme (X39 or A39) X39 indicates mutants Q39E and Q39N A39 indicates an alanine at position 39 Amino acid side-chains and substrates were represented as a simplified outline based on the structure presented in Fig 1 Only noncovalent interactions (dotted lines) involving the residue 39 and E451 were represented 4-OH is represented in the equatorial (4e; pointing towards left) and axial (4a; pointing upwards) positions 3-OH is always in the equatorial position, and a missing 6-OH indicates a 6-deoxy monosaccharide (fucose; third column).

Fig 3 Free energy changes between enzyme–transition state (ES
) complexes formed by Spodoptera frugiperda digestive b-glycosidase (Sfbgly50) mutants at residue 451 ES
complexes are represented as described in Fig 2 X451 indicates mutants E451Q, E451D and E451S A451 indicates an alanine at position 451 For simplification purposes, only part of the E451 side-chain (wild-type) was represented, although in mutants E451Q, E451D and E451S, the interactions geometry might be different Only the noncovalent interactions (dotted lines) involving residue 451 and Q39 were represented Other details are as described in the legend to Fig 2.

(4 and 6) and different active-site residues at positions 39

and 451 (/4e, /4a, /6, g4e, g4a, g6; Tables 5 and 6)

were calculated by isolating each DG
, as described in

the Materials and methods

Interactions involving residue 39

As seen in Fig 1, the Q39 side-chain (Neatom) of the

wild-type Sfbgly50 is a donor in a hydrogen bond with 4-OH

(interactions /4e and /4a) Simultaneously, 4-OH is a

donor in a hydrogen bond with the E451 side-chain

(interaction g4e and g4a) Also, the Q39 side-chain

(Oeatom) is an acceptor in the interaction /3

Two different residues, E and N, were introduced at

position 39, originally occupied by Q Glutamine and

asparagine present similar side-chains, containing hydrogen

bond donor and acceptor atoms (Nd, Ne and Od, Oe,

respectively), in spite of the asparagine side-chain being

shorter [20] Thus, the drastic decrease of the /4e and /4a

energy (68 ± 5% and 78 ± 6%, respectively; Table 5)

may result exclusively from the length increase of these interactions This also indicates that the length decrease of the residue 39 side-chain affects /4e and /4a interactions equally

Replacement of Q39 with E is not a conservative exchange, as the glutamine side-chain is simultaneously a hydrogen bond donor and acceptor, whereas the glutamate side-chain is only a hydrogen bond acceptor [20] Thus, in mutant Q39E, the 4-OH may interact with E451, whereas the E39 side-chain (Oeatom) may form the /3 interaction E39 Oeand Q39 Oeatoms may form similar /3 interactions,

as glutamate Oe and glutamine Oe atoms have similar partial charges ()0.8188e and )0.8086e, respectively) [21] Nevertheless, in mutant Q39E, the replacement of Q with E disrupts the interactions /4e and /4a, because E is not a hydrogen bond donor Considering that the /3 energy was included into /4e and /4a energies during calculations (see the Materials and methods), the values obtained for /4e and /4a (8.9 and 8.2 kJÆmol)1, respectively) when E is at position 39 may actually correspond to the /3 interaction (Table 5)

Based on those estimates of /3 energy, /4e and /4a energy in the wild-type Sfbgly50 should be 10.4 kJÆmol)1 (19.3–8.9) and 5.9 kJÆmol)1(14.1–8.2), respectively (Table 5)

In the ES
complex of the wild-type Sfbgly50, 4-OH has been shown to have a stronger interaction with E451 than with Q39 (g4e¼ 33.2 kJÆmol)1vs /4e¼ 19.3 kJÆmol)1; g4a¼ 18.7 kJÆmol)1vs /4a¼ 14.1 kJÆmol)1) [7] Thus, the esti-mates of /4e and /4a energy presented here (10.4 kJÆmol)1 and 5.9 kJÆmol)1, respectively) show that this energy differ-ence is actually higher than previously calculated, strengthening the importance of E451 in determining Sfbgly50 specificity

The large length of /6 interaction is incompatible with a hydrogen bond In addition, the replacement of Q39 with E (which altered the hydrogen bonding properties of the residue 39 side-chain, while maintaining its length) did not affect the energy of that interaction (Table 5) Moreover, the replacement of Q39 with N, which shortened the length

of the residue 39 side-chain, disrupted /6, as indicated by its negative value (Table 5)

Table 1 Mathematical expressions used to calculate the energy of

noncovalent interactions between residues at positions 39 or 451 and

4-OH or 6-OH in the enzyme–transition state (ES
) complex /, residue

at position 39; g, residue at position 451; 4, glycone hydroxyl 4; 6,

glycone hydroxyl 6; a, axial position; e, equatorial position DG
,

activation energy of the glycosylation step; DDG
, differences in the

activation energy of the glycosylation steps.

Noncovalent interactions

involvingamino acid residues

at position 39

Noncovalent interactions involving amino acid residues

at position 451 Noncovalent

interaction

Expression for

calculation

Noncovalent interaction

Expression for calculation /4e a DDG
– DG

/6 g4e DDG
– DG

g6

a The energy value of /3 is included in these interactions.

Table 2 Steady-state kinetic parameters for hydrolysis of NPbglycosides by the recombinant wild-type Spodoptera frugiperda digestive b-glycosidase (Sfbgly50) (wt) and Sfbgly50 mutants Experiments were carried out at 10 different substrate concentrations and parameters were calculated by using the ENZFITTER software Standard errors were less than 12% of the mean values.

k cat

(s)1)

K m (m M )

k cat /K m (s)1Æm M )1 )

k cat (s)1)

K m (m M )

k cat /K m (s)1Æm M )1 )

k cat (s)1)

K m (m M )

k cat /K m (s)1Æm M )1 )

a Data from [7].

Interactions involving residue 451

In the wild-type Sfbgly50, the 4-OH is a donor in a

hydrogen bond with the E451 side-chain (Oeatom) and an

acceptor of a hydrogen bond with the Q39 side-chain

(Neatom) (Fig 1) Three different residues (Q, D and S)

were introduced at position 451, originally occupied by E

Based on the hydrogen-bonding properties of Q, D and S

side-chains (all of which are hydrogen bond acceptors), they

are also expected to interact with 4-OH

In the mutant E451Q, the residue Q451 side-chain is a

hydrogen bond acceptor (Oeatom) and presents the same

length of E side-chain Thus, the g4e energy values observed

when E and Q are at position 451 (33.2 kJÆmol)1 and

32.2 kJÆmol)1, respectively; Table 6) indicate that this

interaction is not affected by the exchange of a charged

participant (carboxyl group of the E side-chain) for an

uncharged participant (amide group of the Q side-chain)

Otherwise, the replacement of E451 with D and S decreases

the energy of g4e by 36% and 68%, respectively (calculated

from Table 6) As D and S side-chains are shorter than

those of E and Q, the energy decreases probably result from the lengthening of the interaction g4e For the mutant E451S, one cannot rule out that the chemical property changes of the residue 451 side-chain (E to S; polar charged

to polar uncharged) may also have affected the properties of the g4e microenvironment, thus contributing to the observed energy decreases For mutant E451D, as D and

E side-chains are similar, the energy decrease probably results from the change in the length of g4e

Thus, the g4e interactions are closer to an optimum length when either E or Q are at position 451, having the highest possible energy In this situation, the interaction is not affected by the exchange between charged and un-charged participants However, small increments in the interaction length result in energy alteration (as when D is at position 451), whereas larger interaction length (as when S is

at position 451) results in interaction disruption

A similar behavior is observed for the g4a interaction In this case, as the axial 4-OH is positioned farther from the residue 451 side-chain, the length of g4a is always larger than of g4e, resulting in lower energies (Table 6) E451 replacement with Q and D resulted in higher decreases (27% and 57%, respectively) in g4a energy than those observed for g4e (3% and 36%, respectively) (calculated from Table 6), whereas changing E451 for S resulted in similar decreases of g4a and g4e energies (74% and 68%, respectively) This suggests that g4a interaction does not have an optimum length, even when E and Q are at position

451 Consequently, exchange between charged and un-charged participants affects this interaction, and small increments in the interaction length result in high energy decreases

g6 energy has consistently shown negative values, regardless of which residue (E, Q, D and S) is at position

451 (Table 6), indicating that the interaction between 6-OH and any of those residues has consistently been unfavorable

to the formation of ES
This effect, a possible steric hindrance, was not decreased even with the substitution of E451 by amino acids with shorter side-chains, such as D and

S Nevertheless, the replacement of E451 with Q increased that effect, probably owing to the fact that glutamine is bulkier than glutamic acid (114 A˚3 and 109 A˚3, respect-ively) These data suggest that 6-OH and the side-chain of residue 451 are very close in the ES
complex

Table 3 Changes in the transition state energies of the glycosylation

step for different Spodoptera frugiperda digestive b-glycosidase

(Sfbgly50) mutants resulting from deletion of the residue 39 side-chain or

hydroxyl changes in the substrate The comparisons used to calculate

these DDG
values are shown in Fig 2 The DDG
values (representing

the difference in the activation energy of the glycosylation steps) were

calculated using k cat /K m data, as discussed in the Materials and

methods Depending on the column, X39 indicates mutants Q39E or

Q39N A39 indicates mutant Q39A Data for this mutant were

obtained from a previous publication [7].

Comparison

Q39E DDG
(kJÆmol)1)

Q39N DDG
(kJÆmol)1) [X39glc]
fi [X39gal]

[X39gal]
fi [X39fuc]

)9.7 ± 0.1 )14.6 ± 0.2 [X39glc]

fi [A 39 glc]
11.8 ± 0.1 4.1 ± 0.1

[X39gal]
fi [A39gal]

[X39fuc]
fi [A39fuc]

[A39glc]

fi [A39gal]
1.4 ± 0.1 1.4 ± 0.1

[A39gal]

fi [A39fuc]
)1.2 ± 0.2 )1.2 ± 0.2

Table 4 Changes in the transition state energies of the glycosylation step for different Spodoptera frugiperda digestive b-glycosidase (Sfbgly50) mutants resulting from deletion of the residue 451 side-chain or hydroxyl changes in the substrate The comparisons used to calculate the DDG
values (i.e the difference in the activation energy of the glycosylation steps) are shown in Fig 3 The DDG
values were calculated using k cat /K m data, as discussed in the Materials and methods Depending on the column, X451 represents mutants E451Q, E451D or E451S A451 indicates mutant E451A Data for this mutant were obtained from a previous publication [7].

Comparison

E451Q DDG
(kJÆmol)1)

E451D DDG
(kJÆmol)1)

E451S DDG
(kJÆmol)1) [A451glc]

[A451gal]
fi [A451fuc]

[X451glc]
fi [A451glc]

[X451gal]

[X451fuc]

[X451glc]
fi [X451gal]

[X451gal]

fi [X451fuc]

Effect of replacement of residues 451 and 39

on the specificity of Sfbgly50

E451 replacement with Q increased the Sfbgly50 preference

for glucosides to the detriment of galactosides by

five-fold (ðkcat=Km NPbglcÞ=ðkcat=Km NPbgalÞ ¼ 14 for wild-type

Sfbgly50, whereasðkcat=Km NPbglcÞ=ðkcat=Km NPbgalÞ ¼ 75 for

mutant E451Q) (Table 2) This change in specificity was

caused by a significant decrease in the g4a energy

(5.1 kJÆmol)1), whereas g4e was not affected (Table 6)

Thus, the active site of the E451Q mutant is better designed

to stabilize the ES
complex of glucosides (which presents

equatorial 4-OH) than that of the galactosides (which

presents axial 4-OH)

Otherwise, kcat/Km ratios indicate that replacement

of E451 with S decreased the Sfbgly50 preference for

glucosides compared to galactosides by  35-fold (ðkcat=

Km NPbglcÞ=ðkcat=Km NPbgalÞ ¼ 14 for the wild-type Sfbgly50,

whereas ðkcat=Km NPbglcÞ=ðkcat=Km NPbgalÞ ¼ 0.4 for the mutant E451S) (Table 2) This mutation decreased g4e energy by 22.6 kJ mol)1, whereas g4a energy was decreased by 13.9 kJÆmol)1(Table 6), resulting in an active site that is better designed to stabilize the ES
complex of galactosides in comparison to glucosides A previous study showed that the replacement (with a serine) of a glutamate equivalent to E451 in the b-glucosidase (CelB) from

P furiosus (E417), did not change the preference for glucosides vs galactosides [11] Moreover, in contrast to the present study, the mutation E417S had a stronger effect

on Km(increment of 20–100 fold) than on kcat(increment of two-to fourfold)

However, the preference for glucosides vs galactosides was also affected by mutating a residue equivalent to E451

in the Sulfolobus solfataricus b-glycosidase (E432) [12] In this case, the replacement of E432 with C decreased the preference for glucosides by 1.5-fold in comparison to galactosides

The replacement of E451 with D did not significantly modify the preference of Sfbgly50 for glucosides and galactosides (ðkcat=Km NPbglcÞ=ðkcat=Km NPbgalÞ ¼ 14 for wild-type Sfbgly50, whereasðkcat=Km NPbglcÞ=ðkcat=Km NPbgalÞ ¼ 9 for the mutant E451D) (Table 2) because g4e and g4a are equally affected by this mutation, which resulted in a decrease of 11 kJÆmol)1in their energies (Table 6)

In summary, the preference for glucosides was 14-fold higher than for galactosides in the wild-type Sfbgly50 (E at position 451), whereas this ratio decreased to 9 in the E451D mutant and to 1.3 in the E432C mutant of S solfataricus b-glycosidase [12], finally reaching a value of 0.4 in the E451S mutant, in which the preference for galactosides is 2.5-fold higher than for glucosides

It was concluded that modification in the ratio between g4e and g4a energy is a key factor in changing the Sfbgly50 preference for glucosides vs galactosides Moreover, as the length of the residue 451 side-chain determines this ratio, the mutation series Efi D fi S may be useful in changing the preference for glucosides and galactosides of other family

1 b-glycosidases

The preference for fucosides was also affected by the replacement of E451 with other residues Introduction of

Q at this position increased the preference of Sfbgli50 for fucosides by 17-fold compared with galactosides (ðkcat=Km NPbfucÞ=ðkcat=Km NPbgalÞ ¼ 39 for the wild-type Sfbgly50, whereas ðkcat=Km NPbfucÞ=ðkcat=Km NPbgalÞ ¼ 669 for mutant E451Q) (Table 2) This specificity change resulted from an increase in the steric hindrance between the 6-OH and residue 451 (interaction g6; Table 6) As galactosides and fucosides differ only in the 6-OH, which is lacking in fucosides, the ES
complexes formed with galactosides is less stable than that formed with fucosides Introduction of D and S at position 451 had a less significant effect on the Sfbgli50 preference for fuco-sides vs galactofuco-sides (ðkcat=Km NPbfucÞ=ðkcat=Km NPbgalÞ ¼

39 for the wild-type Sfbgly50, whereas ðkcat=Km NPbfucÞ=

ðkcat=Km NPbgalÞ ¼ 108 for mutant E451D and 50 for mutant E451S) (Table 2) because these replacements did not significantly change g6 interaction (Table 6) The effect

on the preference for fucosides by mutation E432C in the

S solfataricus b-glycosidase [12] is similar to that of the mutations E451S and E451D in Sfbgly50, suggesting that a

Table 5 Energies of noncovalent interactions between different residues

at position 39 and glycone hydroxyls 4 and 6 in the enzyme–transition

state (ES
) complex 4, glycone hydroxyl 4; 6, glycone hydroxyl 6; e,

equatorial position; a, axial position; /4e, nonconvalent interaction

between residue 39 and equatorial 4-OH; /4a, nonconvalent

interac-tion between residue 39 and axial 4-OH; /6, noncovalent interacinterac-tion

between residue 39 and 6-OH The results are expressed as DG

(kJÆmol)1) DG
values (activation energy of the glycosylation step)

were calculated referring to the disruption of the interaction, thus

positive values indicate an interaction that stabilizes the ES
complex;

the opposite is true for negative values.

Noncovalent interaction

Amino acid residue at position 39

a

Q is the residue present in wild-type Sfbgly50, data were obtained

from a previous publication [7].bThese values include /3

(inter-action between residue 451 and 3-OH).

Table 6 Energies of noncovalent interactions between different residues

at position 451 and the glycone hydroxyls 4 and 6 4, glycone hydroxyl 4;

6, glycone hydroxyl 6; e, equatorial position; a, axial position; g4e,

noncovalent interaction between residue 451 and equatorial 4-OH;

g4a, noncovalent interaction between residue 451 and axial 4-OH; g6,

noncovalent interaction between residue 451 and 6-OH The results are

expressed as DG
(kJÆmol)1) The DG
values (activation energy of the

glycosylation step) were calculated referring to the disruption of the

interaction, thus positive values indicate an interaction that stabilizes

the enzyme–transition state (ES
) complex; the opposite is true for

negative values.

Noncovalent

interaction

Residue at position 451

g4e 33.2 ± 0.7 32.2 ± 0.5 21.3 ± 0.6 10.6 ± 0.5

g4a 18.7 ± 0.2 13.6 ± 0.2 8.0 ± 0.1 4.8 ± 0.1

g6 )4.4 ± 0.4 )11.5 ± 0.3 )6.9 ± 0.3 )5.0 ± 0.3

a Data from a previous publication [7].

steric hindrance with the 6-OH is also present in the

S solfataricusenzyme Additionally, the mutation E417S in

the P furiosus b-glucosidase increased the rate of hydrolysis

of 6-phospho b-galactosides, indicating that shortening the

residue at position 417 (which is equivalent to E451 in

Sfbgly50) set more room in the active site to the binding of

groups attached to the 6-OH [11] Accordingly, 6-phospho

b-glycosidases have a serine at a position corresponding to

E451 Therefore, steric hindrance between the 6-OH and

E451 seems to be a widespread phenomenon among family

1 b-glycosidases

The Sfbgly50 preference for fucosides is also affected

by replacements at position 39 Indeed the introduction

of N at this position increased by eightfold, the preference

for this substrate (ðkcat=Km NPbfucÞ=ðkcat=Km NPbgalÞ ¼ 39

for the wild-type Sfbgly50, whereas ðkcat=Km NPbfucÞ=

ðkcat=Km NPbgalÞ ¼ 336 for Q39N mutant) (Table 2), a

specificity change resulting from /6 disruption (Table 6)

Thus, the ES
complex with galactosides is destabilized,

whereas a complex with fucosides, which lack 6-OH, is not

affected

In summary, all mutations at position 39 and 451 resulted

in an increase in the preference of Sfbgly50 for fucosides,

because both of these mutations disrupt /6 (an interaction

that stabilizes the ES
complex with galactosides and

glucosides) or increase the steric hindrance with 6-OH

(g6, a interaction that destabilizes the ES
complex with

galactosides and glucosides)

Design of b-glycosidases and modification in specificity

The interaction energies involving substrates and three

different residues (Q, E and N) at position 39 and four

residues at position 451 (E, Q, D and S) in Sfbgly50 were

considered here Using the wild-type and mutant residues at

those positions, we can identify 12 different Sfbgly50 active

sites Six of these mutants – the wild-type enzyme (Q39E451)

and five mutants (E39E451, N39E451, Q39D451, Q39N451

and Q39S451) – had already been characterized, but six

double mutants (E39D451, E39N451, E39S451, N39D451,

N39N451 and N39S451) remain to be studied

However, it is possible to predict the specificity of these

Sfbgly50 double mutants, as long as we assume the tested

noncovalent interactions to remain independent (replacing

one residue does not affect the interactions formed by

another) and ES
complex energy to be determined by the

sum of the energy values of all noncovalent interactions

formed by their active site residues with 4-OH and 6-OH of

different substrates (glucosides, galactosides and fucosides)

The preferred substrate (presenting the highest rate of

hydrolysis) would be that which forms the noncovalent

interactions set with the highest possible energy This

strategy of designing and predicting the specificity of new

Sfbgly50 mutants is currently being tested in our laboratory

Results with Sfbgly50 mutants showed that changes in

enzyme specificity resulted from destabilization of the ES

complex, which caused Sfbgly50 mutants to be less active

than the in wild-type counterparts Similar results were

observed in other studies aiming to change b-glycosidase

specificity through mutagenesis [10–12,22] This suggests

that simultaneous mutations of other residues, perhaps even

outside the active site, are required for a fine tuning of the

active site structure, in order to increase the catalytic activity

of the mutant b-glycosidases However, the identification of these residues is not an easy task owing to the large number

of amino acid residues that require to be tested The utilization of in vitro evolution could help us to solve this problem [8,9,23]

Therefore, the strategy suggested here, to produce efficient b-glycosidases with planned specificity, would be

to perform selection of the appropriate residues at positions

39 and 451, followed by in vitro-directed evolution Acknowledgements

This project is supported by FAPESP (Fundac¸a˜o de Amparo a` Pesquisa

do Estado de Sa˜o Paulo) and CNPq (Conselho Nacional de Desen-volvimento Cientı´fico e Tecnolo´gico) We thank A Wu and R Dillon for critically reading our manuscript S.R.M., W.R.T and C.F are staf members of the Biochemistry Department and research fellows of CNPq E.H.P.A is an undergraduate student and a research fellow

of CNPq.

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Supplementary material The following material is available from http://www blackwellpublishing.com/products/journals/suppmat/EJB/ EJB4354/EJB4354sm.htm

Fig S1 Purfication of the recombinant mutant Sfbgly50