1
Development of Novel Sugar Isomerases by Optimization 1
of Active Sites in Phospho Sugar Isomerases for 2
Monosaccharides 3
4
Soo-Jin Yeom1, Yeong-Su Kim1, and Deok-Kun Oh* 5
6
Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, 7
Republic of Korea 8
Journal section: Biotechnology 9
10
Running title: YEOM ET AL. 11
DEVELOPMENT OF NOVEL SUGAR ISOMERASE 12
13
14
15
*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
20
21
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
23
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
44
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INTRODUCTION 45
46
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
94
MATERIALS AND METHODS 95
96
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
100
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
110
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
131
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
146
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
173
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
182
RESULTS AND DISCUSSION 183
184
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
198
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
226
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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based molecular docking: A case study of CDOCKER-A CHARMm-based MD 375
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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
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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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A C E
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
ffici
ency
100
200
300
ive
cata
lytic
effi
cien
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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A B C
40 100 80
vers
ion
yiel
d (%
)
20
30
nver
sion
yie
ld (%
)
40
60
80
vers
ion
yiel
d (%
)
40
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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