Development of Novel Sugar Isomerases by Optimization of ...Nov 28, 2012 · 2 22 ABSTRACT 23 24...

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Development of Novel Sugar Isomerases by Optimization 1

of Active Sites in Phospho Sugar Isomerases for 2

Monosaccharides 3

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Soo-Jin Yeom1, Yeong-Su Kim1, and Deok-Kun Oh* 5

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Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, 7

Republic of Korea 8

Journal section: Biotechnology 9

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Running title: YEOM ET AL. 11

DEVELOPMENT OF NOVEL SUGAR ISOMERASE 12

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*Corresponding author. Mailing address: Department of Bioscience and Biotechnology, 16

Konkuk University, 1 Hwayang-Dong, Gwangjin-Gu, Seoul 143-701, Republic of 17

Korea. Phone: 82-2-454-3118. Fax: 82-2-444-6176. E-mail: [email protected]. 18

1 These authors contributed equally to this work. 19

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.02539-12 AEM Accepts, published online ahead of print on 30 November 2012

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ABSTRACT 22

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Phospho sugar isomerases can catalyze the isomerization of not only phospho 24

sugar but also of monosaccharides, suggesting that the phospho sugar isomerases 25

can be used as sugar isomerases that do not exist in nature. Determination of active 26

site residues of phospho sugar isomerases, including ribose-5-phosphate isomerase 27

from Clostridium difficile (CDRPI), mannose-6-phosphate isomerase from Bacillus 28

subtilis (BSMPI), and glucose-6-phosphate isomerase from Pyrococcus furiosus 29

(PFGPI), was accomplished by docking of monosaccharides onto the structure 30

models of the isomerases. The determinant residues, including Arg133 of CDRPI, 31

Arg192 of BSMPI, and Thr85 of PFGPI, were subjected to alanine substitutions 32

and found to act as phosphate-binding sites. R133D of CDRPI, R192N of BSMPI, 33

and T85Q of PFGPI displayed the highest catalytic efficiencies for 34

monosaccharides at each position. These residues exhibited 1.8-, 3.5-, and 4.9-fold 35

higher catalytic efficiencies for the monosaccharides compared with the wild-type 36

enzyme, respectively. However, the activities of these 3 variant enzymes for 37

phospho sugars, as the original substrates, disappeared. Thus, R133D of CDRPI, 38

R192N of BSMPI, and T85Q of PFGPI are no longer phospho sugar isomerases; 39

instead, they are changed to a D-ribose isomerase, an L-ribose isomerase, and an L-40

talose isomerase, respectively. In this study, we used substrate-tailored 41

optimization to develop novel sugar isomerases which are not found in nature 42

based on phospho sugar isomerases. 43

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INTRODUCTION 45

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The development of new enzymes has long been a goal in the field of protein 47

engineering, and many advances have been made regarding directed evolution and 48

rational design (1). New enzymes with novel catalytic activities as biocatalysts can 49

facilitate and simplify many chemical processes to produce a broad range of products 50

(2). The protein engineering of enzymes has emerged as a powerful enabling technology 51

for development of a new biocatalyst. Directed evolution does not require structural 52

information but often results in various variants. Moreover, it requires a high-53

throughput screening system and can unpredictably alter enzyme properties. Rational 54

design, employing site-directed mutagenesis, is relatively inexpensive and simple. 55

However, detailed structural knowledge of a protein is often unavailable, and the effects 56

of various mutations can be extremely difficult to predict (1). Substrate-tailored 57

optimization is an easy way to create novel enzymes and combines the advantages of 58

directed evolution and rational design while concurrently removing the aforementioned 59

disadvantages. In substrate-tailored optimization, the target substrate is docked to an 60

enzyme with different function using its determined structure or homology model, and 61

residues of the active site that interact with the substrate are selected and optimized 62

using site-directed mutagenesis. 63

Recently, carbohydrates have attracted attention as cell surface receptors of cells in 64

glycobiology due to their effective functions. Synthesized carbohydrates that disrupt 65

carbohydrate-dependent processes are emerging as important therapeutic agents (3). 66

Among the carbohydrates, monosaccharides are the simplest carbohydrates and the 67

most basic compounds in glycobiology. Currently, monosaccharides are synthesized 68


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using chemical or biological methods, but the chemical method has several 69

disadvantages, including complex purification steps and the formation of by-products 70

and chemical waste. To overcome these disadvantages, monosaccharides are 71

synthesized through microbial and enzymatic reactions using various enzymes (4). Rare 72

monosaccharides have a wide variety of applications, including their uses as low-calorie 73

sweeteners, antioxidants, glycosidase inhibitors, nucleoside analogs, antiviral agents, 74

anticancer agents, and immunosuppressants (5-11). However, natural biosynthetic 75

enzymes are insufficient for the synthesis of various rare monosaccharides, and specific 76

sugar isomerases have not yet been identified in nature. For example, some sugar 77

isomerases such as L-talose isomerase, D-ribose isomerase, D-talose isomerase, L-xylose 78

isomerase, and L-lyxose isomerase have not been identified because organisms do not 79

require such rare monosaccharides to survive. Thus, the discovery of new natural 80

monosaccharide biosynthetic enzymes via screening is very difficult, and such enzymes 81

may be obtained by modifying naturally occurring enzymes by using protein 82

engineering techniques. 83

Three phospho sugar isomerases, namely, ribose-5-phosphate isomerase (RPI) (12), 84

mannose-6-phosphate isomerase (MPI) (13), and glucose-6-phosphate isomerase (GPI) 85

(14), participate in the pentose phosphate pathway and glycolysis metabolism 86

(Supplemental Figure 1). Because these isomerases are involved in the isomerization of 87

phospho sugars, they can also catalyze the isomerization of various monosaccharides 88

owing to their broad substrate specificity (15-19) (Figure 1). 89

In this study, we developed D-ribose isomerase, L-ribose isomerase, and L-talose 90

isomerase, based on RPI from Clostridium difficile (CDRPI), MPI from Bacillus subtilis 91


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(BSMPI), and GPI from Pyrococcus furiosus (PFGPI), respectively, via substrate-92

tailored optimization. 93

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MATERIALS AND METHODS 95

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Materials. Kits for PCR product purification, gel extraction and plasmid preparation, 97

as well as the DNA-modifying enzymes, were purchased from Promega. The phospho 98

sugar and monosaccharide standards were purchased from Sigma and Carbosynth. 99

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Bacterial strains, plasmids and growth conditions. C. difficile ATCC 43255, B. 101

subtilis ATCC 23857, P. furiosus DSM 3638, Escherichia coli ER2566, and plasmid 102

pET-28a (+) were used as the sources of genomic DNA for RPI, MPI, and GPI; as host 103

cells; and as the expression vector, respectively. Recombinant E. coli cells for enzyme 104

expression were cultivated in 500 ml of Luria-Bertani (LB) medium in a 2,000-ml flask 105

containing 20 μg/ml kanamycin at 37 °C with shaking at 250 rpm. When the OD600 of 106

the culture reached 0.6, 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was 107

added to the culture medium and the culture was incubated with shaking at 150 rpm at 108

16 °C for 16 h to express the enzyme. 109

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Cloning and site-directed mutagenesis of phospho sugar isomerases. Primer 111

sequences used for gene cloning were based on the DNA sequence of the CDRPI 112

(GenBank accession number AM180355). Forward (5′-113

TTTCATATGAAGATAGGATTAGGCT-3′) and reverse primers (5′- 114

TTTCTCGAGTTATTTATTATGTTTTTCTTC-3′) were designed to introduce the NdeI 115


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and XhoI restriction sites at the underlined sequences, respectively. Primer sequences 116

used for gene cloning were based on the DNA sequence of BSMPI (GenBank accession 117

number AF324506). Forward (5′-TTTCATATGACGCATCCTTTATT-3′) and reverse 118

primers (5′-TTTCTCGAGTTAAGGATGAGATATCA-3′) were designed for 119

introduction of the NdeI and EcoRI restriction sites at the underlined sequences, 120

respectively. The sequence of the primers used for gene cloning was based on the DNA 121

sequence of the glucose-6-phosphate isomerase from P. furiosus (GenBank accession 122

number AF381250). Forward (5′-TTTCATATGTATAAGGAACCTTTTGGAGTG-3′) 123

and reverse primers (5′-TTTCTCGAGCTACTTTTTCCACCTGGGATTATC-3′) were 124

designed to introduce the NdeI and XhoI restriction sites at the underlined sequences, 125

respectively. 126

Amplified DNA fragments were purified using a PCR purification kit (Promega). The 127

purified sequences were ligated into individual restriction enzyme sites of pET-28a(+). 128

The resulting plasmids were used to transform the E. coli ER2566 strain. Site-directed 129

mutagenesis was performed using the QuikChange kit (Stratagene). 130

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Purification of phospho sugar isomerases. Washed recombinant cells were 132

resuspended in 50 mM phosphate buffer containing 300 mM NaCl, 10 mM imidazole 133

and 0.1 mM PMSF as a protease inhibitor. The resuspended cells were disrupted using 134

ultrasonication with the samples kept on ice. Cell debris was removed by centrifugation 135

at 13,000×g for 20 min at 4 °C, and the supernatant was filtered through a 0.45-μm 136

pore-size filter. The filtrate was applied to a HisTrap HP chromatography column (GE 137

Healthcare) equilibrated with 50 mM phosphate buffer. The column was washed 138

extensively with the same buffer, and the bound protein was eluted with a linear 139


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gradient from 10 to 250 mM imidazole at a flow rate of 1 ml/min. The active fractions 140

were collected and dialyzed at 4 °C for 24 h against 50 mM piperazine-N,N′-bis(2-141

ethanesulfonic acid) (PIPES) buffer (pH 7.0). After dialysis, the resulting solution was 142

used as the purified enzyme. Purification steps using a column were carried out using a 143

fast protein liquid chromatography (FPLC) system (Bio-Rad Laboratories) in a cold 144

room. 145

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Comparative homology modeling. Homology modeling of CDRPI was performed 147

using MODELLER (20) and optimized using FoldX (21) based on the X-ray structure 148

model of RPI from Clostridium thermocellum (PDB code 3HEE) as a template. A 149

homologous search and sequence alignment were conducted using sequence analysis 150

and multiple-sequence alignment modules, respectively. Based on the optimized 151

alignment, 5 comparative models of the target sequence were generated using 152

MODELLER by applying the default building routine “model” with fast refinement. 153

This procedure has an advantage in that the best model can be selected from several 154

candidate models. Furthermore, variability among the models can be used to evaluate 155

modeling reliability. Energy minimization was performed using the consistent valence 156

force field and the Discover program using the steepest descent and conjugated gradient 157

algorithms. The quality of these models was analyzed using PROCHECK (22). 158

159

Ligand docking. Docking of ribose-5-phosphate/L-talose, mannose-6-phosphate/D-160

talose, and glucose-6-phosphate/L-talose were initially accomplished based on the 161

predicted topological binding sites by several algorithms (23). The automated docking 162

was carried out using the CDOCKER program (Accelrys) (24) based on the Merck 163


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molecular force field (MMFF) and AutoDock 4.0 program suite (25). The active site 164

was defined as the collection of amino acid residues enclosed within a sphere of 4.5 Å 165

radius from the center of the docked substrate. The MD-simulated annealing process 166

was performed using a rigid protein and flexible ligand. Ligand-protein interactions 167

were computed from a full force field, and a final minimization step was applied to 168

ligand docking pose. The minimization consisted of 50 steps of the steepest descent 169

followed by up to 200 steps of conjugated-gradient using an energy tolerance of 0.001 170

kcal mol–1. The substrate orientation giving the lowest interaction energy was chosen 171

for additional docking studies. 172

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Analytical methods. The concentrations of phospho sugars and monosaccharides 174

were determined by a Bio-LC system (Dionex ICS-3000) with an electrochemical 175

detector using a CarboPac PAI column. To analyze phospho sugars, the column was 176

eluted at 30 °C with a Na-acetate gradient of 75 mM NaOH and 75 mM NaOH/500 mM 177

Na-acetate. The gradient was increased to 100 mM between 0 and 35 min, to 150 mM 178

between 35 and 38 min, to 350 mM between 38 and 65 min, and then to 500 mM for 75 179

min. The flow rate was 1 ml/min. To analyze monosaccharides, the column was eluted 180

at 30 °C with 200 mM sodium hydroxide at a flow rate of 1 ml/min. 181

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RESULTS AND DISCUSSION 183

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Substrate specificity of phospho sugar isomerases. Three phospho sugar 185

isomerases, including CDRPI, BSMPI, and PFGPI, were cloned and expressed in 186

Escherichia coli and purified as a single band using HisTrap HP affinity 187


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chromatography (15, 17, 18). These wild-type enzymes can catalyze the isomerization 188

reactions not only for phospho sugars but also for monosaccharides. These properties 189

allow these phospho sugar isomerases to be used as candidates for creating new sugar 190

isomerases. The substrate specificity of these enzymes was investigated with the D- and 191

L-forms of the pentoses and hexoses, including talose, allose, mannose, galactose, 192

glucose, altrose, gulose, idose, xylose, arabinose, lyxose, and ribose. Among the 193

monosaccharides, the specific activities of wild-type CDRPI, BSMPI, and PFGPI were 194

the highest for D-ribose, L-ribose, and L-talose, respectively (15, 17, 18) (Table 1). Thus, 195

these phospho sugar isomerases were used in the development of novel sugar 196

isomerases. 197

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Determinant positions at active sites of phospho sugar isomerases for 199

monosaccharides. To identify the determinant residues responsible for developing 200

novel sugar isomerases, we used the crystal structure models of BSMPI (PDB code 201

1QWR), and PFGPI (PDB code 2GC2) and the homology model of CDRPI. The 202

monosaccharides D-ribose, L-ribose, and L-talose were docked onto the phospho sugar 203

isomerases CDRPI, BSMPI, and PFGPI, respectively, using the Surflex docking 204

program (24). Eleven residues of CDRPI, namely, Asp8, His9, Tyr43, Cys66, Thr68, 205

His99, Asn100, Arg110, Arg133, His134, and Arg137; 15 residues of BSMPI, namely, 206

Lys12, Arg14, Trp16, Leu86, Gln95, His97, Lys113, Glu154, Trp117, His172, Leu174, 207

Glu182, Asp188, Tyr191, and Arg192; and 11 residues of PFGPI, namely, Tyr52, 208

Thr71, Thr85, His88, His90, Glu97, Tyr99, His136, Tyr152, His158, and Tyr160 were 209

shown to interact with the docked monosaccharides via hydrogen bonding. These 210

residues were substituted one by one with alanine, and the wild-type and all variant 211


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enzymes were expressed and purified. The activities of the wild-type and variant 212

enzymes were measured using phospho sugars and monosaccharides as substrates 213

(Figure 2). Three variants, CDRPI R133A, BSMPI R192A, and PFGPI T85A, showed 214

the highest activities for D-ribose, L-ribose, and L-talose, respectively. However, the 215

activities of these variants for phospho sugars were negligible. The different activity 216

patterns observed for phospho sugars and monosaccharides indicate that Arg133 of 217

CDRPI, Arg192 of BSMPI, and Thr85 of PFGPI may be molecular determinants that 218

can be used to develop novel sugar isomerases. These residues in phospho sugar 219

isomerases located near the phosphate group of phospho sugar consist of several (more 220

than two) positively charged or polar amino acids (Figure 3A, 3C, and 3E), whereas 221

residues located near the terminal (5 or 6)-OH of the monosaccharide are not typically 222

positively charged or polar amino acids (Figure 3B, 3D, and 3F). Thus, the phosphate-223

binding site of phospho sugar isomerases may contain crucial residues that when 224

substituted with other amino acids result in the creation of a new sugar isomerases. 225

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Development of novel sugar isomerases using site-directed mutagenesis at 227

determinant positions of phospho sugar isomerases. The amino acid residues at 228

determinant positions of phospho sugar isomerases were replaced with other amino 229

acids, including Asp, Gln, Lys, Glu, Tyr, and Ile at position 133 of CDRPI; Glu, Lys, 230

Leu, Asn, and Tyr at position 192 of BSMPI; and Ser, Gln, Asp, and Lys at position 85 231

of PFGPI. Expression of the wild-type and variant enzymes was confirmed by sodium 232

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (data not shown). 233

R133D of CDRPI, R192N of BSMPI, and T85Q of PFGPI displayed the highest 234

catalytic efficiencies for monosaccharides as substrates among the wild-type and variant 235


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enzymes at position 133 of CDRPI, position 192 of BSMPI, and position 85 of PFGPI, 236

respectively (Figure 4). These enzymes exhibited 1.8-, 3.5-, and 4.9-fold higher 237

catalytic efficiencies compared with the corresponding wild-type enzymes, respectively 238

(Table 2). However, the variants showed no activity for phospho sugars as original 239

substrates. Indeed, the variants did not convert phospho sugars into their corresponding 240

products. Monosaccharide production rates for the variant enzymes were higher than 241

those obtained using the wild-type enzymes (Figure 5). 242

Specifically, authentic substrates of the phospho sugar isomerase variants R133D of 243

CDRPI, R192N of BSMPI, and T85Q of PFGPI, were converted from phospho sugars 244

to monosaccharides. These variants are no longer a RPI, a MPI, and a GPI, respectively; 245

instead, they have been changed into a D-ribose isomerase, an L-ribose isomerase, and 246

an L-talose isomerase, respectively, which do not exist in nature. These novel enzymes 247

can contribute rare monosaccharides production. Therefore, novel isomerases were 248

developed based on phospho sugar isomerases via substrate-tailored optimization. 249

L-Ribose has been used as a starting material of L-nucleoside-based pharmaceuticals 250

(26) and potent anti-viral agents for hepatitis B virus and Epstein-Barr virus (27). Its 251

chemical derivatives involve the inhibition of the viral nucleoside synthesis-replication 252

process by exploiting the minor difference in the nucleoside synthesis process between 253

a normal cell and a virus. L-Talofuranosyladenine, an L-talose nucleoside derivative, can 254

be used as a slowly reacting substrate for calf intestinal adenosine deaminase and an 255

inhibitor for the growth of leukemia cells in vitro (28). D-Ribose has been used as a 256

precursor in the synthesis of nucleotide flavor enhancers and riboflavin (vitamin B2) 257

(29). Enzymes that can be used in the biosynthesis of these monosaccharides should be 258

developed. While L-talose isomerase and D-ribose isomerase have not been reported, 259


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one L-ribose isomerase has been described (30). However, this L-ribose isomerase 260

exhibited low activity and no extensive homology with MPI. Thus, the phosphate-261

binding site variant of MPI described above is a new type of efficient L-ribose isomerase. 262

Recently, we applied the phosphate-binding site variant of MPI from Thermus 263

thermophilus (TTMPI R142N) to produce L-ribose, and the enzyme exhibited the 264

highest activity and productivity for L-ribose production ever reported (31). This 265

enzymatic method is superior to the chemical synthetic method presently used in the 266

manufacturing process due to a higher productivity. 267

The substrate specificity of TTMPI was similar to BSMPI. The catalytic efficiencies 268

of TTMPI and its R142N variant (134 and 174 mM−1s−1) for L-ribose were higher than 269

those of BSMPI and its variant R192N (13 and 46 mM−1s−1), whereas the increase of the 270

catalytic efficiency by mutation of BSMPI was higher than that by mutation of TTMPI. 271

TTMPI was used in the previously study for increasing L-ribose production (31), 272

whereas BSMPI were used in this study for the investigation for the general role of 273

phosphate binding residues in the phospho sugar isomerases. 274

275

Structural analysis of novel sugar isomerases. When ribose-5-phosphate, mannose-276

6-phosphate, and glucose-6-phosphate were docked to CDRPI, BSMPI, and PFGPI, 277

respectively, Arg133, Arg192, and Thr85 interacted directly with the phosphate groups 278

of the phospho sugars (Figure 3A, 3C, and 3E). The phosphate group is located at the 279

end of the monosaccharide moiety and may be critical for defining the substrate 280

specificity of the corresponding phospho sugar isomerase. When monosaccharides were 281

docked to modeled structures, phosphate-binding residues in the phospho sugar 282

isomerases did not interact tightly with the terminal hydroxyl groups of the 283


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monosaccharides. Thus, the phosphate-binding sites of the phospho sugar isomerases 284

were molecular determinants with different catalytic activities for phospho sugars and 285

monosaccharides. Furthermore, optimization was accomplished by these sites with other 286

amino acids to develop novel sugar isomerases. 287

When the monosaccharide substrates were docked to the active site pockets of 288

phospho sugar isomerases by ligand docking study, distances from the terminal 289

hydroxyl of the monosaccharides D-ribose, L-ribose, and L-talose to the side chains of 290

CDRPI R133D (2.67 Å) (1 Å = 0.1 nm), BSMPI R192N (2.38 Å), and PFGPI T85Q 291

(2.26 Å) variant enzymes were shorter than those of the respective wild-type enzymes 292

(4.74 Å, 3.85 Å, and 4.83 Å, respectively) (Figure 3B, 3D, and 3F). Therefore, we 293

suggest that these shorter distances between the phosphate-binding sites and terminal 294

hydroxyl groups of monosaccharides may explain the enhanced kcat/Km values obtained 295

for monosaccharide substrates compared with the wild-type enzymes. The variant 296

enzymes exhibited higher activities for other monosaccharides than the wild-type 297

enzymes (Table 1). However, the actual structure of these wild-type and variant 298

enzymes complexes with substrates must be obtained to provide further evidence for 299

these identifications. 300

In summary, new sugar isomerases for the biosynthesis of monosaccharides were 301

developed from phospho sugar isomerases by substrate-tailored optimization method. 302

Each of these new sugar isomerases dissipated the authentic function of phospho sugar 303

isomerases and reinforced catalytic activity for monosaccharide biosynthesis. The 304

crystal structures and homology models in complex with phospho sugars and 305

monosaccharides should allow exploration of how altering the enzyme affects the 306

catalytic properties of the protein at the molecular level. Our findings may be used for 307


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the enzymatic synthesis of chemicals not found in nature, and may be applied to the 308

establishment of new enzymes from naturally occurring enzymes. 309

310

ACKNOWLEDGMENTS 311

This study was funded by the Basic Research Lab. (No. 2010-0019306) Program 312

funded by the National Research Foundation of Korea (NRF) grant, Republic of Korea. 313

314


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REFERENCES 315

316

1. Bornscheuer, UT, Pohl M. 2001. Improved biocatalysts by directed evolution and 317

rational protein design. Curr. Opin. Chem. Biol. 5:137-143. 318

2. Hohne, M, Schatzle S, Jochens H, Robins K, Bornscheuer UT. 2010. Rational 319

assignment of key motifs for function guides in silico enzyme identification. Nat. 320

Chem. Biol. 6:807-813. 321

3. Bertozzi, CR, Kiessling LL. 2001. Chemical glycobiology. Science 291:2357-322

2364. 323

4. Granstrom, TB, Takata G, Tokuda M, Izumori K. 2004. Izumoring: a novel and 324

complete strategy for bioproduction of rare sugars. J. Biosci. Bioeng. 97:89-94. 325

5. Muniruzzaman, S, Pan YT, Zeng Y, Atkins B, Izumori K, Elbein AD. 1996. 326

Inhibition of glycoprotein processing by L-fructose and L-xylulose. Glycobiology 327

6:795-803. 328

6. Livesey, G, Brown JC. 1996. D-Tagatose is a bulk sweetener with zero energy 329

determined in rats. J. Nutr. 126:1601-1609. 330

7. Matsuo, T, Suzuki H, Hashiguchi M, Izumori K. 2002. D-Psicose is a rare sugar 331

that provides no energy to growing rats. J. Nutr. Sci. Vitaminol. (Tokyo) 48:77-80. 332

8. Hossain, MA, Wakabayashi H, Goda F, Kobayashi S, Maeba T, Maeta H. 2000. 333

Effect of the immunosuppressants FK506 and D-allose on allogenic orthotopic liver 334

transplantation in rats. Transplant Proc. 32:2021-2023. 335

9. Levin, GV. 2002. Tagatose, the new GRAS sweetener and health product. J Med 336

Food 5:23-36. 337

10. Levin, GV, Zehner LR, Saunders JP, Beadle JR. 1995. Sugar substitutes: their 338


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

16

energy values, bulk characteristics, and potential health benefits. Am. J. Clin. Nutr. 339

62:1161S-1168S. 340

11. Doong, SL, Tsai CH, Schinazi RF, Liotta DC, Cheng YC. 1991. Inhibition of the 341

replication of hepatitis B virus in vitro by 2',3'-dideoxy-3'-thiacytidine and related 342

analogues. Proc. Natl. Acad. Sci. U. S. A. 88:8495-8499. 343

12. Dickens, F, Williamson DH. 1956. Pentose phosphate isomerase and epimerase 344

from animal tissues. Biochem. J. 64:567-578. 345

13. Rose, IA, O'Connell EL, Schray KJ. 1973. Mannose 6-phosphate: anomeric form 346

used by phosphomannose isomerase and its 1-epimerization by phosphoglucose 347

isomerase. J. Biol. Chem. 248:2232-2234. 348

14. Walker, JI, Faik P, Morgan MJ. 1990. Characterization of the 5' end of the gene 349

for human glucose phosphate isomerase (GPI). Genomics 7:638-643. 350

15. Yeom, SJ, Kim BN, Park CS, Oh DK. 2010. Substrate specificity of ribose-5-351

phosphate isomerases from Clostridium difficile and Thermotoga maritima. 352

Biotechnol. Lett. 32:829-835. 353

16. Park, HY, Park CS, Kim HJ, Oh DK. 2007. Substrate specificity of a galactose 6-354

phosphate isomerase from Lactococcus lactis that produces D-allose from D-psicose. 355

J. Biotechnol. 132:88-95. 356

17. Yoon, RY, Yeom SJ, Park CS, Oh DK. 2009. Substrate specificity of a glucose-6-357

phosphate isomerase from Pyrococcus furiosus for monosaccharides. Appl. 358

Microbiol. Biotechnol. 83:295-303. 359

18. Yeom, SJ, Ji JH, Kim NH, Park CS, Oh DK. 2009. Substrate specificity of a 360

mannose-6-phosphate isomerase from Bacillus subtilis and its application in the 361

production of L-ribose. Appl. Environ. Microbiol. 75:4705-4710. 362


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

17

19. Yoon, RY, Yeom SJ, Kim HJ, Oh DK. 2009. Novel substrates of a ribose-5-363

phosphate isomerase from Clostridium thermocellum. J. Biotechnol. 139:26-32. 364

20. Sali, A, Blundell TL. 1993. Comparative protein modelling by satisfaction of 365

spatial restraints. J. Mol. Biol. 234:779-815. 366

21. Schymkowitz, J, Borg J, Stricher F, Nys R, Rousseau F, Serrano L. 2005. The 367

FoldX web server: an online force field. Nucleic Acids Res 33:W382-8. 368

22. Laskowski, RA, Macarthur MW, Moss DS, Thornton JM. 1993. PROCHECK: a 369

program to check the stereochemical quality of protein structures. J. Appl. Cryst. 370

26:283-291. 371

23. Huang, B, Schroeder M. 2008. Using protein binding site prediction to improve 372

protein docking. Gene 422:14-21. 373

24. Wu, G, Robertson DH, Brooks CL 3rd, Vieth M. 2003. Detailed analysis of grid-374

based molecular docking: A case study of CDOCKER-A CHARMm-based MD 375

docking algorithm. J. Comput. Chem. 24:1549-1562. 376

25. Huey, R, Morris GM, Olson AJ, Goodsell DS. 2007. A semiempirical free energy 377

force field with charge-based desolvation. J. Comput. Chem. 28:1145-1152. 378

26. Okano, K. 2009. Synthesis and pharmaceutical application of L-ribose. Tetrahedron 379

65:1937-1949. 380

27. Ma, T, Pai SB, Zhu YL, Lin JS, Shanmuganathan K, Du J, Wang C, Kim H, 381

Newton MG, Cheng YC, Chu CK. 1996. Structure--activity relationships of 1-(2-382

deoxy-2-fluoro-beta-L-arabinofuranosyl)pyrimidine nucleosides as anti-hepatitis B 383

virus agents. J. Med. Chem. 39:2835-2843. 384

28. Lerner, LM, Mennitt G. 1994. A new synthesis of L-talose and preparation of its 385

adenine nucleosides. Carbohydr Res 259:191-200. 386


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29. De Wulf, P, Vandamme EJ. 1997. Production of D-ribose by fermentation. Appl. 387

Microbiol. Biotechnol. 48:141-148. 388

30. Mizanur, RM, Takata G, Izumori K. 2001. Cloning and characterization of a 389

novel gene encoding L-ribose isomerase from Acinetobacter sp. strain DL-28 in 390

Escherichia coli. Biochim. Biophys. Acta 1521:141-145. 391

31. Yeom, SJ, Seo ES, Kim BN, Kim YS, Oh DK. 2011. Characterization of a 392

mannose-6-phosphate isomerase from Thermus thermophilus and increased L-ribose 393

production by its R142N mutant. Appl. Environ. Microbiol. 77:762-767. 394

395

396

397


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List of Figures 398

399

Fig. 1. Schematic diagrams of reactions catalyzed by phospho sugar isomerases, 400

including CDRPI, BSMPI, and PFGPI. (A) Isomerization between ribose-5-401

phosphate and ribulose-5-phosphate, and between D-ribose and D-ribulose catalyzed by 402

ribose-5-phosphate isomerase from Clostridium difficile (CDRPI). (B) Isomerization 403

between mannose-6-phosphate and fructose-6-phosphate, and between L-ribose and L-404

ribulose catalyzed by mannose-6-phosphate isomerase from Bacillus subtilis (BSMPI). 405

(C) Isomerization between glucose-6-phosphate and fructose-6-phosphate, and between 406

L-talose and L-tagatose catalyzed by glucose-6-phosphate isomerase from Pyrococcus 407

furiosus (PFGPI). 408

409

Fig. 2. Relative catalytic efficiencies of the wild-type and variant enzymes of 410

CDRPI, BSMPI, and PFGPI for phospho sugars and monosaccharides. (A) Relative 411

activities of the wild-type and variant enzymes of CDRPI for ribose-5-phosphate and D-412

ribose. The relative catalytic efficiencies of 100% for ribose-5-phosphate and D-ribose 413

were 500 and 0.6 mM–1 s–1, respectively. (B) Relative activities of the wild-type and 414

variant enzymes of BSMPI for mannose-6-phosphate and L-ribose. The relative catalytic 415

efficiencies of 100% for mannose-6-phosphate and L-ribose were 2014 and 13 mM–1 s–1, 416

respectively. (C) Relative activities of the wild-type and variant enzymes of PFGPI for 417

glucose-6-phosphate and L-talose. The relative catalytic efficiencies of 100% for 418

glucose-6-phosphate and L-talose were 2284 and 3.6 mM–1 s–1, respectively. The black 419

and white bars represent relative activities for phospho sugar and monosaccharide, 420

respectively. The data represent the means of three separate experiments, and the error 421


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bars represent standard deviation. 422

423

Fig. 3. Active site structures of wild-type enzymes of CDRPI, BSMPI, and 424

PFGPI with phospho sugars and monosaccharides. (A) Active site of CDRPI with 425

ribose-5-phosphate. Arg133 (cyan) in CDRPI directly interacted with the phosphate 426

group (red) in the phospho sugar. The dotted line indicates an interaction between the 427

phosphate group of ribose-5-phosphate and the phosphate-binding site of CDPRI. (B) 428

Active site of CDRPI with D-ribose as a substrate. Arg133 (cyan) and Asp132 (magenta) 429

are visible at the bottom of the image. (C) Active site of BSMPI with mannose-6-430

phosphate. The charcoal sphere represents a metal ion. Arg192 (cyan) in BSMPI 431

directly interacted with the phosphate group (red) in the phospho sugar. The dotted line 432

indicates an interaction between the phosphate group of mannose-6-phosphate and the 433

phosphate-binding site of BSMPI. (D) Active site of BSMPI with L-ribose as a substrate. 434

Arg192 (cyan) and Asn192 (magenta) are visible at the bottom of the image. (E) Active 435

site of PFGPI with glucose-6-phosphate. The charcoal sphere represents metal ion. 436

Thr85 (cyan) in PFGPI directly interacted with the phosphate group (red) in the 437

phospho sugar. The dotted line indicates an interaction between the phosphate group of 438

glucose-6-phosphate and the phosphate-binding site of PFGPI. (F) Active site of PFGPI 439

with L-talose as a substrate. Thr85 (cyan) and Gln85 (magenta) are visible at the bottom 440

of the image. The residue, metal ion, and distance are represented as a stick model, 441

sphere, and dashed line, respectively. Docking of phospho sugars and monosaccharides 442

were initially accomplished based on the predicted topological binding sites by several 443

algorithms using homology model of CDRPI and crystal structure of BSMPI and PFGPI. 444

The automated docking was carried out using the CDOCKER program (Accelrys) based 445


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on the Merck molecular force field (MMFF) and AutoDock 4.0 program suite. 446

PROCHECK examination of the mutant enzymes did not show any molecular clashes 447

for the variant side chains. 448

449

Fig. 4. Relative catalytic efficiencies of the wild-type and variant enzymes of 450

CDRPI, BSMPI, and PFGPI for phospho sugars and monosaccharides. (A) Relative 451

activities of the wild-type and variant enzymes of CDRPI for ribose-5-phosphate and D-452

ribose. The relative catalytic efficiencies of 100% for ribose-5-phosphate and D-ribose 453

were 500 and 0.6 mM–1 s–1, respectively. (B) Relative activities of the wild-type and 454

variant enzymes of BSMPI for mannose-6-phosphate and L-ribose. The relative catalytic 455

efficiencies of 100% for mannose-6-phosphate and L-ribose were 2014 and 13 mM–1 s–1, 456

respectively. (C) Relative activities of the wild-type and variant enzymes of PFGPI for 457

glucose-6-phosphate and L-talose. The relative catalytic efficiencies of 100% for 458

glucose-6-phosphate and L-talose were 2284 and 3.6 mM–1 s–1, respectively. The black 459

and white bars represent relative activities for phospho sugar and monosaccharide, 460

respectively. The data represent the means of three separate experiments, and the error 461

bars represent standard deviation. 462

463

Fig. 5. Production of phospho sugars and monosaccharides by the wild-type and 464

variant enzymes of CDRPI, BSMPI and PFGPI. (A) Production of D-ribulose (open 465

symbol) from D-ribose and of ribulose-5-phosphate (closed symbol) from ribose-5-466

phosphate by the wild-type (circle) and R132D variant (square) CDRPIs. (B) 467

Production of L-ribose (open symbol) from L-ribulose and of fructose 6-phosphate 468

(closed symbol) from mannose 6-phosphate by the wild-type (circle) and R192N variant 469


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(square) BSMPIs. (C) Production of L-tagatose (open symbol) from L-talose and of 470

fructose 6-phosphate (closed symbol) from glucose 6-phosphate by the wild-type (circle) 471

and T85Q variant (square) PFGPIs. The data represent the means of three separate 472

experiments, and the error bars represent standard deviations. 473


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A

B

C

Fig. 1


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A B C

(%)

100

120

(%)

120

140

(%) 140

160

ve c

atal

ytic

effi

cien

cy (

40

60

80

ve c

atal

ytic

effi

cien

cy (

40

60

80

100

e ca

taly

tic e

ffici

ency

(

60

80

100

120

CDRPI

Wild

D8A

H9A

Y43A

C66A

T68A

H99A

N100

AR1

10A �

H134

AR1

37A

Rel

ativ

0

20

R133

A

BSMPI

WildK1

2AR1

4AW1

6AL8

6AQ9

5AH9

7AK1

13AE1

15A

W117

AH1

72AL1

74AE1

82AD1

88AY1

91A �

Rel

ativ

0

20

R192

AR

elat

ive

0

20

40

Wild

Y52A

T85AT7

1AH9

0AE9

7AY9

9AH1

36AY1

52AH1

58A

H88A

Y160

A

PFGPI

Fig. 2


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B D F

Fig. 3


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A B C

y (%

)

200

y (%

)

400

(%)

500

lativ

e ca

taly

tic e

ffici

ency

50

100

150

lativ

e ca

taly

tic e

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ency

100

200

300

ive

cata

lytic

effi

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cy

200

300

400

CDRPI

Wild

R133

DR1

33A

R133

QR1

33K

R133

ER1

33Y

R133

I

Re

0

BSMPI

Wild

R192

AR1

92E

R192

KR1

92L

R192

NR1

92Y

Rel

0

Wild T85A

T85S

T85Q

T85D

T85K

Rel

ati

0

100

PFGPI

Fig. 4


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40 100 80

vers

ion

yiel

d (%

)

20

30

nver

sion

yie

ld (%

)

40

60

80

vers

ion

yiel

d (%

)

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60

Time (min)

0 10 20 30 40 50 60

Con

v

0

10

Time (min)

0 10 20 30 40

Con

0

20

Time (min)

0 10 20 30 40 50 60

Con

v0

20

Fig. 5


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TABLE 1. Relative activities of the wild-type and variant enzymes of CDRPI, BSMPI,

and PFGPI for monosaccharides

aThe relative activities of 100% for CTRPI for D-ribose, BSMPI for L-ribose, and PFGPI

for L-talose were 7.4, 22.5, and 0.6 μmol min–1 mg–1, respectively.

n.d., not detected

The data represent the means and standard deviations of three separate experiments.

Substrate

Relative activity (%)a

CDRPI BSMPI PFGPI

Wild-Type R132D Wild-Type R192N Wild-Type T85Q

D-Talose 1±0.1 2±0.2 54±1.3 95±2.4 45±1.5 152±1.2 L-Talose 100±2.5 156±2.3 2±0.1 5±0.2 100±0.7 456±4.3 D-Allose 18±0.2 31±1.5 1±0.1 2±0.1 71±1.5 260±12 L-Allose 8±0.2 15±0.8 15±0.9 35±0.1 51±1.4 192±7.2 D-Mannose n.d.b n.d. 19±0.5 42±0.7 28±0.2 128±1.5 L-Mannose n.d. n.d. 3±0.1 6±0.1 31±0.3 135±6.8 D-Galactose n.d. n.d. n.d. n.d. 3±0.1 15±0.2 L-Galactose n.d. n.d. n.d. n.d. 4±0.1 19±0.3 D-Glucose n.d. n.d. n.d. n.d. 34±0.8 142±5.6 L-Glucose n.d. n.d. n.d. n.d. 41±1.1 150±9.5 D-Altrose n.d. n.d. n.d. n.d. 18±0.2 75±1.3 L-Altrose n.d. n.d. n.d. n.d. 11±0.1 39±0.5 D-Gulose n.d. n.d. n.d. n.d. 33±0.2 139±2.7 L-Gulose n.d. n.d. n.d. n.d. 26±0.4 122±4.5 D-Idose n.d. n.d. n.d. n.d. 33±0.2 138±4.6 L-Idose n.d. n.d. n.d. n.d. 33±0.3 139±8.7 D-Xylose n.d. n.d. n.d. n.d. 37±0.1 148±8.1 L-Xylose n.d. n.d. n.d. n.d. 39±1.5 148±3.6 D-Arabinose n.d. n.d. n.d. n.d. 24±0.4 118±1.8 L-Arabinose n.d. n.d. n.d. n.d. 14±0.1 60±1.7 D-Lyxose n.d. n.d. 62±0.2 99±3.7 28±0.6 113±2.1 L-Lyxose n.d. n.d. 1±0.1 2±0.1 31±1.2 129±3.8 D-Ribose 79±0.9 116±3.4 2±0.1 3±0.1 88±0.5 290±15 L-Ribose 4±0.1 5±0.2 100±1.1 257±6.0 63±1.1 248±14


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TABLE 2. Kinetic parameters of the wild-type and variant enzymes at position 132 of

CDRPI for D-ribose, at position 192 of BSMPI for L-ribose, and at 85 position of PFGPI

for L-talose.

Enzymes Km (mM) kcat (s–1) kcat/Km (mM–1 s–1)

CDRPI Wild-type 245±10 139±8 0.56±0.04

R132A 217±4 132±3 0.61±0.02

R132I 320±31 71±5 0.22±0.03

R132Q 204±11 106±3 0.52±0.03

R132K 265±4 149±3 0.56±0.01

R132E 217±0.4 148±1 0.68±0.005

R132Y 292±5 161±1 0.41±0.005

R132D 216±5 214±3 0.99±0.03

BSMPI Wild-type 688±13 9095±91 13.2±0.3

R192A 722±43 4653±113 6.5±0.4

R192N 569±27 26113±886 45.9±2.7

R192K 792±4 7348±47 9.3±0.08

R192E 789±61 6331±259 17.6±1.0

R192L 590±12 6293±45 11.0±0.2

R192Y 998±44 17595±670 17.6±1.0

PFGPI Wild-type 133±4.9 475±7 3.6±0.1

T85A 186±5.6 960±24 5.2±0.2

T85S 146±3.6 381±5 2.6±0.1

T85Q 100±2.5 1756±22 17.6±0.5

T85D 185±4.7 448±7 2.4±0.1

T85K 205±6.1 396±14 1.9±0.1 The data represent the means and standard deviations of three separate experiments.


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