Enhanced Catalytic Efficiency in Quercetin-4â²-glucoside Hydrolysis

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Enhanced catalytic efficiency in quercetin-4´-glucoside hydrolysis of Thermotoga maritima #-glucosidase A by site-directed mutagenesis Huihui Sun, Yemin Xue, and Yufei Lin J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 16 Jun 2014 Downloaded from http://pubs.acs.org on June 21, 2014

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Journal of Agricultural and Food Chemistry

Enhanced catalytic efficiency in quercetin-4´-glucoside

1 2

hydrolysis of Thermotoga maritima β-glucosidase A by

3

site-directed mutagenesis huihui sun1,2, yemin xue1*, yufei lin1

4 5 6 7 8

1

Department of Food Science and Nutrition, GinLing College, Nanjing Normal

University, Nanjing, PR China 210097 2

College of Life Science, Nanjing Normal University, Nanjing, PR China

210046

9 10 11

∗

Corresponding authors (Phone: +86-25-85794123, Fax: +86-25-83598901,

E-mail addresses: [email protected])

12 13 14 15 16 17 18 19 20 21 22 23 24 25 1

ACS Paragon Plus Environment


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Abstract Te-BglA and Tm-BglA are glycoside hydrolase family 1 β-glucosidases

27

from

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respectively, with 53% sequence identity. However, Te-BglA could more effectively

29

hydrolysed isoflavone glucosides to their aglycones than could Tm-BglA, possibly

30

due to the difference in amino acid residues around their glycone binding pockets.

31

Site-directed mutagenesis was used to replace the amino acid residues of Tm-BglA

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with the corresponding residues of Te-BglA, generating three single mutants (F221L,

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N223L and G224T), as well as the corresponding three double mutants

34

(F221L/N223L,

35

(F221L/N223L/G224T). The seven mutants have been purified, characterized and

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compared to the wild-type Tm-BglA. The effects of the mutations on kinetics, enzyme

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activity, and substrate specificity were determined. All mutants showed pH-activity

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curves narrower on the basic side and wider on the acid side, and had similar optimal

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pH and stability at pH 6.5 to 8.3. They were more stable up to 85°C, but G224T

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display higher optimal temperature than Tm-BglA. Seven mutants indicated an

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obvious increase in catalytic efficiency toward p-nitrophenyl β-D-glucopyranoside

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(pNPG) but an increase or not change in Km. All mutants showed a decrease in

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catalytic efficiency of isoflavonoid glycosides, and were not change for F221L and

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lost for N223L in enzymatic hydrolysis on quercetin-glucosides. Contrarily, G224T

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resulted in a dramatic increase conversion of Q4´ (35.5%) and Q3,4´ (28.6 %) in

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accord with an increased turnover number (kcat, 1.4 ×) and catalytic efficiency (kcat/Km,

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2.2 ×) as well as a decrease in Km (0.24) for Q4'. Modeling showed that G224T

Thermoanaerobacter

ethanolicus

F221L/G224T and

JW200

and

N223L/G224T)

Thermotoga

and

2


one

maritima,

triple

mutant

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mutation at position 224 may enhance the interaction between G224T and 5-OH and

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3-OH on the quercetin back-bone of Q4'.

50 51

Introduction

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The flavonoids are polyphenolic compounds which are found abundantly in

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plants and may play a dietary role in reducing the risk from chronic diseases such as

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cardiovascular disease and cancer. Flavonoids are divided into several types according

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to the structure, such as flavanones, flavonols, isoflavones and so on (1, 2). However,

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flavonoids are generally not found as free aglycones, but rather as complex conjugates

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with sugar residues (3), and the aglycone is likely to have a greater biological effect

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than the glycoside (4). Generally isoflavone aglycones possess higher pharmaceutical

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activity than isoflavone glycosides. Therefore, the abilities of many bacterial and

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fungal β-glucosidases for converting isoflavone glycosides into the aglycones have

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been extensively studied in recent year. (5-8). Many thermostable β-glucosidases from

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Sulfolobus solfataricus, Pyrococcus furiosus, Thermoanaerobacter ethanolicus and

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Thermotoga maritima classified under family 1 of the glycoside hydrolases (GH1) are

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flavonoid-hydrolysing enzymes. It have shown that different capable of hydrolyzing

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isoflavone glycosides in different GH1 β-glucosidase (e.g. the specific activity of

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β-glucosidase from Sulfolobus solfataricus for isoflavones was: daidzin > glycitin >

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genistin, those from P. furiosus followed the order genistin > daidzin > glycitin) (5, 6).

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Our previous work have also shown that the GH1 β-glucosidase (Te-BglA) from the

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Thermoanaerobacter ethanolicus JW200, could more effectively hydrolysed

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isoflavone glucosides of soy flour to their aglycones than could Tm-BglA from T.

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maritima (7). There are differences in hydrolysis of flavones glycosides by these 3



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enzymes, reflecting the diversity of their specificity towards flavones glycosides.

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However, our knowledge of the molecular determinants of aglycone specificity in

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β-glucosidases remains limited. A promising bio-catalysis requires engineering to

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function as a topic of considerable industrial interest. Obtaining a high hydrolysis of

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glucosylated flavonoids is dependent on a high aglycone specificity of the enzyme

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that has been the topic of previous studies (9-12). Therefore, defining the factors that

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govern the fundamental difference in hydrolyzing flavonoids between the different

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β-glucosidases will help unravel the details of the catalytic mechanism in order to find

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residues that influence specificity of the enzyme.

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β-Glycosidases grouped in GH1 are active upon a broad range of substrates,

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share the same tertiary structure, a (β/α)8 barrel with the conserved glutamate residues

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at the carboxy-terminal ends of strands 4 and 7 serving as the catalytic acid/base and

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catalytic nucleophile (13,14). The monosaccharide forming the non-reducing end of

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the substrate binds to subsite −1 (or glycone subsite) and the remaining part of the

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substrate is accommodated in the aglycone binding region, which may be formed by

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several sub-sites (+1,+2, +3 and so on) (15-18). The -1 subsite of various

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β-glycosidases have been studied (16), and have shown that the glycone specificity is

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determined by a hydrogen network formed between glycone hydroxyls and amino

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acid residues of the sub-site, and the amino acid residues close to the aglycone

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binding sub-sites +1 varied with various β-glucosidases in GH1 (Figure 1). Based on

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the sequence alignment of family 1 enzymes and analysis of the TmBglA structural

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model, three residues (F221, N223 and G224) in the active site pocket located on

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β-strand 5 close to sugar binding sub-site +1 were selected for mutagenesis to

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investigate the influence on the enzyme kinetics (using the substrate p-nitrophenyl

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β-D-glucopyranoside (pNPG)), thermostability and pH profiles and the hydrolysis of 4


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flavonoids glycosides (soy isoflavone glycosides and quercetin glucosides) in order to

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modulate the specificity of β-glucosidases that have valuable biotechnological

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applications.

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MATERIALS AND METHODS

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Strains, plasmids, and chemicals

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The p-nitrophenyl (pNP) glycoside substrate pNP-β-D-glucopyranoside (pNPG)

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as well as Isoflavone standards of daidzin (Din), daidzein, genistin (Gin), and

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genistein

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High-performance liquid chromatography (HPLC) grade methanol and acetonitrile

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were purchased from Fisher Scientific (Hanover Park, IL). Quercetin glucosides

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standards of quercetin-3,4'-O-glucose

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quercetin-4'-O-glucose (Q4'), quercetin (Q) were purchased from Extrasynthese

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Chemical Co. (France). Escherichia coli JM109 (Promega, Madison, WI) was used as

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host for the expression of β-glucosidase A gene from T. maritima, via plasmid pHsh

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(19). DNA-modifying enzymes and polymerases were purchased from TAKARA

113

(Dailian, China). All oligonucleotide primers were synthesized by Sangon (Shanghai,

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China). All other chemicals used were analytical grade reagents unless otherwise

115

stated. Recombinant enzyme was induced by a temperature shift from 30 to 42°C. E.

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coli was grown in Luria-Bertani (LB) supplemented with 100 µg ampicillin ml-1.

were

purchased

from

Sigma

Chemical

Co.

Louis,

MO).

(Q3,4'), quercetin-3-O-glucose (Q3),

117 118

(St.

Sequence alignment and molecular docking

5



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The sequences of Tm-BglA and other GH1 β-glycosidases were aligned using

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ClustalW 2.0.12 and ESPript (20). The structure of β-D-cellotetraose was docked into

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the active site of Tm-BglA (PDB code 2WC4) using AutoDock version 4.2 (21). The

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coordinate files of both protein and ligand required for docking calculation were

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prepared by AutoDockTools. The non-polar hydrogens were deleted by the program,

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and the partial charges were merged to the carbon atoms. The sugar substrates were

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treated as rigid, and the rotatable bonds were set automatically by the program. The

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protein portion was set as flexible receptor by assigning the catalytic residues (E165

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and E351) as flexible residues. AutoGrid was performed to pre-calculate the grid map

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of interaction energy prior to docking. The grid size was set at 60 × 60 × 60 with a

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grid point spacing of 0.258 Å at the center of protein. A Lamarckian genetic algorithm

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was used with a population size of 300, maximum number of energy evaluations of

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2500000, maximum number of generations of 25000 and uniform crossover mode.

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Other parameters were set as default. The docked conformation was visualized using

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Accelrys DS Visualizer 3.0 (22).

134 135

Cloning and site-directed mutagenesis

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Plasmid constructions were carried out according to standard procedures (23).

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The T. maritima β-glucosidase A gene, Tm-bglA, based upon that reported in Genbank

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entry X74163, was amplified with pET-20b-Tm-bglA as a template (7, 8), and ligated

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at restriction sites XbaI and XhoI with pHsh (19), resulting recombinant plasmid was

140

named as pHsh-Tm-bglA. Mutagenesis was performed in order to introduce the 6


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following designed changes F221L, N223L, G224T, F221L/N223L, F221L/G224T,

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N223L/G224T, F221L/N223L/G224T, respectively. Eight oligonucleotides for each

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mutation were designed to contain the corresponding nucleotide changes (see Table

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1). These oligonucleotides and pHsh-Tm-bglA as template were used to introduce

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mutations using PCR under the following conditions: one cycle of denaturation at

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95°C for 5 min, 30 cycles of denaturation at 94°C for 40 s, annealing at 55°C for 40 s,

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extension at 72°C for 4 min, and extra extension at 72°C for 7 min. The PCR products

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were purified using the QIAquick PCR purification kit, and phosphated with T4

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polynucleotide kinase, and ligated into the expression vector pHsh (19), resulting in

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the expression vectors containing exchange mutant Tm-BglA. The nucleotide changes

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were then sequenced by Biological Services Unit of Shang Hai.

152 153

Expression and purification

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To unambiguously characterize the activity of the mutational protein, the

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recombinant protein was purified to homogeneity as follows. E. coli JM109

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containing pHsh-Tm-bglA and mutational plasmid were grown in LB media at 30°C,

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pH 7.0 containing 100 µg ampicillin ml-1 to an OD600 of 0.7-0.8, and then transferred

158

into a 42°C shaking incubator, and grown for a further 9 h at 42 °C. The cells were

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harvested by centrifugation, and washed twice with water, then resuspended in

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binding buffer (5 mM imidazole, 0.5 M NaCl, and 20 mM Tris–HCl, pH 7.9). After

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sonication and centrifugation (30 min, 9,600 ×g, 4°C), the supernatant was heated for

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30 min at 70°C to remove most of the host cell proteins by centrifugation. The 7



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resulting supernatants were purified by Ni-affinity chromatography (Novagen) to

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homogeneity as determined by the sodium dodecyl sulfate polyacrylamide gel

165

electrophoresis (SDS–PAGE) using 12% polyacrylamide running gels with 4%

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polyacrylamide stacking gels. Protein concentration was determined by the Bradford

167

method (24).

168 169

Enzyme assays

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Enzyme activity was quantified by p-nitrophenol (pNP) release from

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p-nitrophenyl β-D-glucopyranoside (pNPG). The reactions were performed in 50 mM

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potassium phthalate buffer (PPB, pH 6.2) containing 2 mM pNPG and 10 µl of

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suitably diluted enzyme for 5 min at 90°C, the reaction was stopped by the addition of

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600 µL of 1 M Na2CO3. The color that developed was read at 405 nm and translated

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to micromoles of pNP using a standard graph prepared under the same conditions.

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One unit was determined as the amount of enzyme producing 1 µmol of pNP/min

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under the above assay conditions. All reactions were done in triplicate.

178 179

Determiation of Kinetic parameters

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For determinations of the kinetic parameters (Km, Vmax kcat/Km) of pNPG,

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reaction was carried out at the optimum conditions using 0.02 to 0.2 mM pNPG.

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Kinetic parameters, Km and Vmax, were determined by the Lineweaver-Burk

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representation of the Michaelis-Menten model. Each experiment was done in

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duplicate, and measurements were made in triplicate. The standard error was recorded 8


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Enhanced Catalytic Efficiency in Quercetin-4â²-glucoside Hydrolysis

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