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Carbohydrate Epimerase の触媒作用, 構造, その応用例

Carbohydrate Epimerase の触媒作用, 構造, その応用例

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J. Appl. Glycosci., 57,1―6 (2010) !C 2010 The Japanese Society of Applied Glycoscience Review Catalysis, Structures, and Applications of Epimerases

(Received July 10, 2009; Accepted August 23, 2009)

Susumu Ito1,＊

1Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus (1, Senbaru, Nishihara-cho, Okinawa 903 ―0213, Japan)

Abstract: More than 20 types of carbohydrate epimerase have been reported to date. Distinctly different en- zymes recognize the C1, C2, C3, C4, C5 or C6 position of carbohydrate substrates. They include, for instance, 1-epimerase, N-acetyl-D-glucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, 2- epimerase, D--5-phosphate 3-epimerase, L-ribulose-5-phosphate 4-epimerase, UDP- 4- epimerase, dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase, GDP- 3,5-epimerase and ADP-L-glycero-D- mannoheptose 6-epimerase. Their biological properties, catalytic mechanisms and tertiary structures are very diverse. This review focuses on the catalysis, structures and applications of carbohydrate epimerases that have recently been characterized in detail.

Key words: carbohydrate epimerase, catalytic property, reaction mechanism, application

Metabolisms of proceed via many kinds glucosamine 2-epimerase (EC 5.1.3.8), UDP-N -acetyl- of which catalyze oxidation, reduction, dehydra- glucosamine 2-epimerase (EC 5.1.3.14), cellobiose 2-epi- tion, acetylation and epimerization. Carbohydrates fulfill merase (EC 5.1.3.11), D-ribulose-5-phosphate 3-epimerase many biological roles in all living cells, such as energy (EC 5.1.3.1), L-ribulose-5-phosphate 4-epimerase (EC source, structural elements and molecular recognition 5.1.3.4), UDP-galactose 4-epimerase (EC 5.1.3.2), dTDP- markers and are used as precursors for the biosynthesis of 6-deoxy-D-xylo-4-hexulose 3,5-epimerase (EC 5.1.3.13), cellular building blocks.1) GDP-mannose 3,5-epimerase (EC 5.1.3.18) and ADP-L- Epimerase is a member of isomerases, which catalyze glycero-D-mannoheptose 6-epimerase (EC 5.1.3.20). Ac- the inversion of stereochemistry in biological (or syn- cording to Allard et al .,1) the reaction mechanism of car- thetic) molecules. Carbohydrate epimerases catalyze the bohydrate epimerases can be divided into four types, reversible interconversion of carbohydrates.1,2) There are which involve 1) transient keto intermediate (UDP- many reports on the enzymatic properties, genes, location galactose 4-epimerase and ADP-L-glycero-D-manno- in metabolic pathways and sources of carbohydrate epi- 6-epimerase), 2) protonation／deprotonation (GDP- merases (http:／／www.expasy.org／enzyme／; http:／／brenda mannose 3,5-epimerase), 3) nucleotide elimination and re- -enzymes.org／; http:／／www.find-health-articles.com／msh_ addition (UDP-N-acetylglucosamine 2-epimerase)or4)C- 20-carbohydrate-epimerases.htm). The enzymes have been C bond cleavage (L-ribulose-5-phosphate 4-epimerase). targeted for antimicrobial drug designs in order to create For these several years, detailed tertiary structures and／or new agents against infectious diseases in humans.1,3―6) reaction mechanisms of carbohydrate epimerases have However, there have been few reports on commercial ap- been reported in the literature. plications of carbohydrate epimerases to date. In this re- view, I focus on recent topics concerning the enzymes 1. N-Acetyl-D-glucosamine 2-epimerase (AGE). with respect to their catalytic properties and structures and In bacteria, some orthologs in the genomes of photo- to production of bioactive mono- and synthetic cyanobacteia have been annotated as AGE. The and bioethanol. tertiary structure of an AGE from Anabaena sp. CH1 (aAGE; PDB code 2gz6) was solved and shown to be a

Catalytic properties and structures of carbohydrate homodimer having an (α／α)6 barrel core structure by Lee epimerases. et al .7) (see Fig. 2). Together with the microenvironment More than 20 types of carbohydrate epimerase have around active-site cleft, the residues Arg57, His239, Glu been reported to date. Usually, the C1, C2, C3, C4, C5 242, Glu308, His372 and Arg375 were shown to be re- and C6 positions of free or substituted mono- and oligo- quired for activity by site-directed mutagenesis experi- saccharides are recognized by distinctly different enzymes, ments. The reversible epimerization at the C2 position of such as aldose 1-epimerase (EC 5.1.3.3), N-acetyl-D- GlcNAc is mediated by the conserved residues His239 (deprotonation) and His372 (protonation) as the acid／base ＊ Corresponding author (Tel.／Fax. +81―98―895―8804, E-mail: sito catalysts. @agr.u-ryukyu.ac.jp). In mammalian cells, the physiological role of AGE had Abbreviations: AGE, N-acetyl-D-glucosamine 2-epimerase; CE, been suggested to be the regulation of the renin- cellobiose 2 epimerase; RaCE, CE from Ruminococcus albus; - angiotension system because high activity was found in RmlC, dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase; DTE, D- 3-epimerase; DPE, D-psicose 3-epimerase; aAGE, AGE pigs, rats and humans, and the porcine kidney 8) from Anabaena sp.; Neu5A, N-acetylneuraminic acid. had renin-binding activity. However, other studies sug- 2 J. Appl. Glycosci., Vol. 57, No. 1 (2010) gest that AGE functions primarily as the enzyme for the or mannose moiety of cello-oligosaccharides, , β- conversion of ManNAc to GlcNAc and plays a key role mannobiose (4-O-β-D-mannopyranosyl-D-mannose) and in sialic acid .9,10) globotriose [O-α-D-galactopyranosyl-(1→4)-O-β-D-galacto- pyranosyl-(1→4)-D-]. The products from lactose, 2. Cellobiose 2-epimerase (CE). β-mannobiose and globotriose are epilactose (4-O- CE from Ruminococcus albus (RaCE) has also been β-D-galctopyranosyl-D-mannose), 4-O-β-D-mannopyranosyl- suggested to have a basal core structure of an (α／α)6 bar- D-glucose and O -α-D-galactopyranosyl-(1→4)-O -β-D- rel, as judged by the secondary structure prediction and galactopyranosyl-(1→4)-D-mannose, respectively (see structure-based alignment with AGEs.11) The residues Arg Fig. 3). The products from cello-oligosaccharides were as- 52, Phe114, His243, Glu246, Trp249, Trp303, Trp304, signed to the corresponding 2-epimerized products. The Glu308, His374 and Arg377 were determined to be re- equilibrium ratio between lactose and epilactose was ap- quired for activity by site-directed mutagenesis,12) as proximately 60:40. Cellobitol, lactitol, N-acetyl-D- shown in Fig. 1. Like in aAGE, His243 and His374 were glucosamine, N-acetyl-D-mannosamine and N-acetyl- assigned to serve as the active site acid／base catalysts be- lactosamine did not serve as substrates. At present, func- cause RaCE has no activity around pH 5. This mechanis- tional CEs have been reported to occur only in bacte- 11,13,15,16) tic proposal is supported by the pKa value of His (around ria, and their physiological role(s) has not been 6.3) in comparison with those of Glu and Asp (around clarified yet. They share less than 20% identity to aAGE,7) 4.1). CE reactions may also involve a protonation／depro- and no homology was observed with the other hetero- tonation step. gonous epimerases. CE was first found in the culture fluid of the anaerobic ruminal bacterium, R. albus 7 (ATCC 27210T).13) It cata- 3. Mannose 2-epimerase activity. lyzes the reversible epimerization of cellobiose to 4-O -β- Centeno et al .17) reported that CmEpiA, formerly desig- D-glucopyranosyl-D-mannose. Recently, we purified RaCE nated Unk2 (AAS19694) of unknown function in the from R. albus NE1 cells and sequenced the encoding Cellvibrio mixtus genome, had a weak epimerization ac- gene for the first time.11,14) We also reported the gene se- tivity at the C2 position of mannose, generating glucose. quences and catalytic properties of CEs from Eubacterium The four possible catalytic residues are well conserved as cellulosolvens 15) and Bacteroides fragilis cells.16) Unlike Arg63, His256, Glu324 and His389. However, the phylo- AGE, the CEs appear to exist as a monomeric form under genetic position of CmEpiA is on a branch different from defined conditions. They catalyzed a hydroxyl stereoisom- those of CEs.16) CE can 2-epimerize β-mannobiose but has erism at the C2 positions of the reducing terminal glucose no activity toward mannose.

4. dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase (RmlC). RmlC epimerizes both the C3′and C5′positions of dTDP-6-deoxy-D-xylo-4-hexulose , yielding dTDP-6- deoxy-L-lyxo-4-hexulose. The enzyme in Streptococcus suis (PDB code 2ixl) requires a conserved His as the base and a conserved Tyr as the acid.18) Recent study with the RmlC from Pseudomonas aeruginosa (PDB code 2ixi); clarifies that the enzyme has a double-stranded β-helix structure, as shown in Fig. 2, and uses the conserved Tyr 134 and His65 as the acid／base catalysts, where the epimerization at C5′proceed faster than that at C3′.4) L- is commonly found in glycoconjugates of bacteria, including pathogenic ones, and neither the nor the enzymes required for the rhamnose biosynthesis are found in humans. RmlC is involved in the dTDP-L- rhamnose biosynthetic pathway that converts glucose-1- phosphate to dTDP-L-rhamnose.19)

5. D-Tagatose 3-epimerase (DTE) and D-psicose 3- epimerase (DPE). DTE catalyzes the reversible epimerization of various ketohexoses at the C3 position, and many DTEs have Fig. 1. A model structure and possible catalytic amino-acid resi- been characterized from various sources (AAL45542, AL dues of the R. albus CE (RaCE). 93912b, BAB50266, BX294153, F72381, NC_005126, The model structure of RaCE was constructed with porcine kid- NP_435986, NP_865388). DTE family enzymes catalyze 12) ney AGE (PDB code 1fp3) as a template. In the ribbon stereodia- the interconversion of D- to D-psicose. D-Psicose gram representation of an (α／α)6 barrel core structure (red, α- helices; cyan and green, sheets and loops), the amino acid resi- can be synthesized from D-fructose by the enzymes from β- 20) dues possibly involved in catalysis are shown in the CPK represen- strains of Pseudomonas cichorii and Rhodobacter tation (upper and lower). sphearoides.21) The tertiary structure of Mn2+-dependent Carbohydrate Epimerase 3

Fig. 2. The tertiary structures of typical carbohydrate epimerases. The structures were searched for at the Worldwide Protein Data Bank (http:／／www.wwpdb.org／) and incorporated into the figure. M2, dimmer; M4, tetramer; M5, pentamer.

DTE from P. cichorii has been shown to be a dimeric form of a (β／α)8 core structure (TIM-barrel fold; PDB code 2qul), as shown in Fig. 2, and Mn2+ ion is coordi- nated with Glu152, Asp185, His211 and Glu246 in the active-site cleft.20) The Glu152 and Glu246 residues appear to involve in the deprotonation／protonation at the C3 po- sition of D-tagatose. Although the Zn2+-dependent D- ribulose-5-phosphate 3-epimerase, a key enzyme in the Calvin cycle, differs greatly in sequence identity (14%) from DTE, the reaction mechanism analogous to DTE is suggested to be adopted to this epimerase having a TIM- barrel fold (PDB code 1tqx).3) Recently, the gene for a putative DTE in the Agrobac- terium tumefaciens genome was cloned and expressed in Escherichia coli cells.22) The recombinant enzyme had very high kcat and catalytic efficiency (kcat／Km) toward D- psicose relative to D-tagatose, so that the A. tumefaciens enzyme was re-named DPE. The DPE exhibits high se- quence similarities of 54―71% to DTE family enzymes from various microorganisms, but not to other epimerases. Fig. 3. Structures of epimerized saccharides described in this arti- cle. The activity of DPE is enhanced greatly in the presence of Mn2+ ions, a characteristic distinct from those of DTE and other known epimerases. The tertiary structure of charides have numerous applications in the pharmaceuti- DPE from A. tumefaciens (PDB code 2hk1) has been cal, food and agricultural industries, especially as thera- solved by Kim et al .23) (Fig. 2). The DPE is a homo- peutics, functional foods and prebiotics.24,25) Commercial tetramer and each monomer is a variation of the classical epimerases have been extracted mainly from microorgan- TIM-barrel fold. The metal-binding site is coordinated isms and used for experimental reagents so far. Hereafter, with Glu150, Asp138, His209, Glu244 and two water we describe the recent contribution of some carbohydrate molecules. Based on the proximity of Glu150 and Glu244 epimerases, which synthesize rare and valuable mono- and to the C3 atom of the bound substrate D-fructose, it was oligosaccharides, to these industrial fields, as shown in speculated that the deprotonation (forward) is achieved by Fig. 3. Glu244 and protonation (reverse) is initiated by Glu150 at the C3 epimerization center. In fact, mutant proteins with 1. N-Acetylneuraminic acid (Neu5A). Glu150Gln and Glu244Gln completely lost their activity. Neu5A exhibits versatile, biological functions as a re- ceptor for microbes, viruses, toxins and hormones and as Production and biological activities of epimerized a regulator of the immune system.26) Neu5A is produced products. on an industrial scale from GlcNAc and pyruvate via Studies on mono- and oligosaccharides production by ManNAc by combination of recombinant porcine kidney microbial enzymes have been promoted because rare sac- AGE and a Neu5A lyase.27) Because the mammalian AGE 4 J. Appl. Glycosci., Vol. 57, No. 1 (2010) requires ATP and Mg2+ ions for activity,28) the use of D-frucose with overall yield of 25% for each reaction was cyanobacteria AGE should have more potential for the in- archived by Menavuvu et al ., using the immobilized en- dustrial production of Neu5A. zyme reactor system.44)

2. Epilactose. 5. D-Tagatose. Epilactose is formed from cow milk by heat and alkali D-Tagatose has attained the generally recognized as safe treatments.29,30) A considerable amount of epilactose is also (GRAS) status under U.S. FDA and is efficacious for use contained in a prebiotic, .31) We showed that CEs as a low-calorie, full-bulk sweetener in foods, beverages, can synthesize epilactose directly from lactose.14) By inter- health foods and dietary supplements.45) It is approxi- vention tests with rats, epilactose has been found to pos- mately 90% as sweet as , supplies 1.5 kcal／gof sess versatile health-promoting properties, which include energy (as compared to 4 kcal／g from sucrose), and thus stimulation of bifidobacteria growth, facilitation of min- is currently in clinical trial as a new drug for treating type eral absorption, reduction of plasma non-high-density cho- 2 diabetes.46) lesterols and suppression of the conversion of primary bile D-Tagatose can be synthesized from D- by a acids to carcinogenetic secondary bile acids.14,32,33) CE has DPE from A. tumefaciens 22) and a DTE from P. cicho- the advantage because it can directly produce epilactose rii.20,47) However, D-sorbose is not inexpensive for the in- from lactose without byproducts in comparison with re- dustrial production of D-tagatose. The use of D-fructose as ported chemical synthesis of epilactose.30,34) There are substrate by immobilized L- isomerase (EC many reports that epilactose is dietary-experienced for hu- 5.3.1.4) from Geobacillus stearothermophilus appears mans; a considerable amount of epilactose is detected in much more promising.48) Commercially available D- heat-sterilized milk, dairy products and lactulose. CE is tagatose is chemically synthesized from lactose, which is expected to increase the value of lactose, cheese whey and first hydrolyzed to glucose and galactose. The galactose milk by creating novel milk products with prebiotic prop- formed is isomerized under alkaline conditions to D- erties. tagatose by calcium hydroxide.

3. D-Psicose. Application of epimerase to biomass-to-ethanol pro- D-Psicose (D-ribo-2-hexulose or D-allulose) is a C3 epi- cess. mer of D-fructose found in processed cane and beet mo- Bioethanol is becoming an attractive alternative to fos- lasses, and food products, for example, coffee, corn sil fuels. For future sustainable and economical production snacks, seasoning sauce and dried fruits.35) By baking, a D- of bioethanol, plant biomass is an attractive feedstock, psicose-containing butter cookie has recently been shown rather than edible feedstock (corn and other grains, to gain antioxidant activity due to the browning reaction.36) and tubers). Cellulosic biomass is a complex D-Psicose can lower plasma glucose levels and reduce mixture of carbohydrate polymers, and its hydrolysates body fat accumulation in rats, suggesting suppression of contain glucose, galactose, mannose, D- and L- the hepatic lipogenic activity37) and may also be of thera- peutic value in the treatment of diseases such as athero- sclerosis.38) As a potent anthelmintic, D-psicose has been shown to inhibit the motility, growth and reproductive maturity of Caenorhabditis elegans.39) D-Psicose can be produced from D-fructose by the ac- tions of a DTE from P. cichorii,20) a DPE from A. tumefa- ciensis 22) and a DTE from R. sphaeroides,21) which exhib- ited the highest substrate specificity toward D-tagatose, D- psicose and D-fructose, respectively. The equilibrium ra- tios between D-psicose and D-fructose of the three were approximately 20:80, 30:70 and 20:80, respectively.

4. D-. The unusual D-allose has shown to exhibit a potent in- hibitory effect on the reactive oxygen species generated from opsonized -stimulated neutrophilis.40) D- Allose inhibits the proliferation of some cancer cell lines in vitro.41) D-Allose induces G1 cell cycle arrest but not apoptosis and significantly up-regulates the gene expres- Fig. 4. The catabolic pathway of L-arabinose in engineered S. cer- sion of thioredoxin-interacting protein, which is often sup- evisiae cells. 42) 43) pressed in tumor cells. Bhuiyan et al . demonstrated L-Arabinose is incorporated into S. cerevisiae by its own galac- the concept of production of D-allose from D-fructose by tose permease Gal2 (GAL2). It is then converted to D--5- immobilized DTE and L-rhamnose isomerase. On the phosphate by a series of the L. plantarum enzymes, AraA (araA), AraB (araB )andAraD(araD ), expressed in the cells. D Xylulose laboratory scale, the reaction yield was 20% and 40% for - - 5-phosphate is an intermediate of the phosphate pathway the conversion of D-fructose to D-psicose and D-psicose to and is directed to the glycolytic pathway under anaerobic condi- D-allose, respectively. Mass production of D-allose from tions, finally producing ethanol. Carbohydrate Epimerase 5 arabinose. The ideal ethanol fermenter is Saccharomyces Special Coordination Funds for Promoting Science and Technology, cerevisiae. However, one of serious problems of the MEXT (Ministry of Education, Culture, Sports, Science and Tech- nology, Japan). I sincerely thank all the members for promoting and pentose-to-ethanol process is that the yeast can not fer- contributing to this project. ment and utilize pentose . Many attempts have been made to establish a D-xylose- REFERENCES fermenting pathway in S. cerevisiae by metabolic (ge- 49,50) netic) engineering. Fortunately, S. cerevisiae galactose 1 ) S.T.M. Allard, M.F. Giraud and J.H. Naismith: Epimerases: permease (Gal2), corresponding to the GAL2 gene, can structure, function and mechanism. Cell. Mol. Life Sci., 58, transport L-arabinose. Wisselink et al .51) succeeded in fer- 1650―1665 (2001). 2 ) R.A. Field and J.H. Naismith: Structural and mechanistic basis menting ethanol from L arabinose by S. cerevisiae after - of bacterial sugar nucleotide-modifying enzymes. Biochemis- evolutionary engineering for improved of try, 42, 7637―7647 (2003). this pentose. The yield of ethanol reached 0.43 g／gofL- 3 ) J. Caruthers, J. Bosch, F. Buchner, W.V. Voorhis et al .: Struc- arabinose after a 70-h fermentation. They introduced an ture of a ribulose 5-phosphate 3-pimerase from Plasmodium falciparum. Proteins, 62, 338―342 (2006). expression vector, harboring araD (AraD, L-ribulose-5- 4 ) C. Dong, L.L. Major, V. Srikannathasan, J.C. Errey et al .: phosphate 4-epimerase) together with araA (AraA, L- RmlC, a C3′and C5′carbohydrate epimerase, appears to oper- arabinose isomerase) and araB (AraB, L-ribulokinase) ate via an intermediate with an unusual twist boat conversion. from Lactobacillus plantarum, into S. cerevisiae cells, as J. Mol. Biol ., 365, 146―159 (2007). 5 ) R. Shi, M. Pineda, E. Ajamian, Q. Cui et al : Structure of L- illustrated in Fig. 4. More recently, they reported that, af- xylulose 3-epimerase (UlaE) from the anaerobic L-ascorbate ter selection of an evolved S. cerevisiae showing im- utilization pathway of Escherichia coli: identification of a proved rates of consumption of , complete fer- novel phosphate binding motif within a TIM barrel fold. J. Bacteriol ., 190, 8137―8144 (2008). mentation of a mixture containing 3% (w／v) D-glucose, 6 ) B. Pacheco, M. Maccarana, D.R. Goodlett, A. Malmström and 1.5% (w／v) D-xylose and 1.5% (w／v) L-arabinose by the L. Malmström: Identification of the active site of DS- yeast was achieved.52) This is the first case of a large-scale epimerase 1 and requirement of N-glycosylation for enzyme function. J. Biol. Chem., 284, 1741―1747 (2009). biomass-to-ethanol process using carbohydrate epimerase. 7 ) Y.C. Lee, H.M. Wu, Y.N. Chang, W.C. Wang and W.H. Hsu: The central cavity from the (alpha／alpha)6 barrel structure of Prospects for future progress. Anabaena sp. CH1 N-acetyl-D-glucosamine 2-epimerase. J. Carbohydrate epimerases reported to date are very di- Mol. Biol ., 367, 895―908 (2007). 8 ) L. Luska, M. Krause, K. Danker, W. Reutter and R. verse biochemically and catalytically. The recent reports Horstkorte: Primary structure and expression analysis of hu- indicate that catalytic events of many epimerases can be man UDP-N-acety-glucosamine-2-epimerase ／ N-acetylmanno- explained by the protonation／deprotonation mechanism. samine kinase, the bifunctional enzyme in bio- To finalize this mechanism proposed for most epimerases, thynthesis. FEBS Lett., 454, 341―344 (1999). 9 ) C. Schmitz, M. Gotthardt, S. Hinderlich, J.R. Leheste et al .: it is required to further identify the critical roles of their Normal blood pressure and plasma renin activity in mice lack- catalytic residues and conformational changes of interme- ing the renin-binding protein, a cellular renin inhibitor. J. Biol. diates involved in the reactions. The tertiary structures of Chem., 275, 15357―15362 (2000). 10) S.J. Luchansky, K.J. Yarema, S. Takahashi and C.R. Bertozzi: different classes of carbohydrate epimerases are also very GlcNAc 2-epimerase can serve a catabolic role in sialic acid different, as shown in Fig. 2. These results suggest that metabolism. J. Biol. Chem., 278, 8035―8042 (2003). they have evolved from different ancestral genes in the 11) S. Ito, S. Hamada, K. Yamaguchi, S. Umene et al .: Cloning line of evolution, which may reflect high divergence at and sequencing of the cellobiose 2-epimerase gene from an obligatory anaerobe, Ruminococcus albus. Biochem. Biophys. their primary sequence and structure levels. Res. Commun., 360, 640―645 (2007). Postgenomic study is very useful for elucidation of the 12) S. Ito, S. Hamada, H. Ito, H. Matsui et al .: Site-directed evolutional history of current proteins. For instance, func- mutagenesis of possible catalytic residues of cellobiose 2- epimerase from Ruminococcus albus. Biotechnol. Lett., 31, tional CE and CE-like proteins have been suggested to 1065―1071 (2009). distribute widely in the microbial genomes of various gen- 13) T.R. Tyler and J.M. Leatherwood: Epimerization of disaccha- era in the phyla Bacteroidetes, Firmicutes and Proteobac- rides by enzyme preparations from Ruminococcus albus. Arch. teria.16) Unidentified and useful carbohydrate epimerases Biochem. Biophys., 119, 363―367 (1967). 14) S. Ito, H. Taguchi, S. Hamada, S. Kawauchi et al .: Enzymatic would therefore be found in microbial genomes that have properties of cellobiose 2-epimerase from Ruminococcus albus not yet been sequenced. Metagenomic approaches will and the synthesis of rare oligosaccharides by the enzyme. also allow us to discover diverse carbohydrate epimerases. Appl. Microbiol. Biotechnol ., 79, 433―441 (2008). 15) H. Taguchi, T. Senoura, S. Hamada, H. Matsui et al .: Cloning In this review, I briefly described metabolic engineering and sequencing of the gene for cellobiose 2-epimerase from a of whole yeast cells for the biomass-to-ethanol process. ruminal strain of Eubacterium cellulosolvens. FEMS Microbiol. Metabolic engineering is a method to improve or generate Lett., 287,34―40 (2008). new metabolic reactions or whole pathways in microor- 16) T. Senoura, H. Taguchi, S. Ito, S. Hamada et al .: Identifica- tion of the cellobiose 2-epimerase gene in the genome of Bac- ganisms. This approach would be promising and make it teroides fragilis NCTC 9343. Biosci. Biotechnol. Biochem., possible to produce commercially valuable, complex oli- 73, 400―406 (2009). gosaccharides without extracting a series of the involved 17) M.S.J. Centeno, C.I.P.D. Guerreiro, F.M.V. Dias, C. Morlan et al .: Galactomannan hydrolysis and mannose metabolism in enzymes from cells. Many microbial cells are expected to Cellvibrio mixtus. FEMS Microbiol. Lett., 261, 123―132 possess a variety of mechanisms for the synthesis of (2006). epimerized products from either natural or chemical sub- 18) C.J. Dong, L.L. Major, A. Allen, W. Blankenfeldt et al .: strates or both. High-resolution structures of RmlC from Streptococcus suis in complex with substrate analogs locate the active site of this class of enzyme. Structure, 11, 715―723 (2003). Part of our studies appearing in this review was supported by the 6 J. Appl. Glycosci., Vol. 57, No. 1 (2010)

19) A. Melo and L. Glaser: The mechanism of 6-deoxyhexose syn- oxygen species from neutrophils. J. Biosci. Bioeng., 96,89―91 thesis. II. Conversion of deoxythymidine diphosphate 4-keto-6- (2003). deoxy-D-glucose to deoxythymidine diphosphate L-rhamnose. 41) L. Sui, Y. Dong, Y. Watanabe, F. Yamaguchi et al .: The in- J. Biol. Chem., 243, 1475―1478 (1968). hibitory effect and possible machanisms of D-allose on cancer 20) H. Yoshida, M. Yamada, T. Nishitani, G. Takada et al .: Crys- cell proliferation. Int. J. Oncol ., 27, 907―912 (2005). tal structures of D-tagatose 3-epimerase from Pseudomonas 42) F. Yamaguchi, M. Takata, K. Kamitori, M. Nonaka et al .: cichorii and its complexes with D-tagatose and D-fructose. J. Rare sugar D-allose induces specific up-regulation of TXNIP Mol. Biol ., 374, 443―453 (2007). and subsequent G1 cell cycle arrest in hepatocellular carci- 21) L. Zhang, W. Mu, B. Jiang and T. Zhang: Characterization of noma cells by stabilization of p27kip1. Int. J. Oncol ., 32, 377― D-tagatose-3-epimerase from Rhodobacter sphaeroides that 385 (2008). converts D-fructose into D-psicose. Biotechnol. Lett., DOI 43) S.H. Bhuiyan, Y. Itami, Y. Rokui, T. Katayama and K. Izu- 10.1007／s10529-009-9942-3 (2009). mori: D-Allose production from D-psicose using immobilized 22) H.J. Kim, E.K. Hyun, Y.S. Kim, Y.J. Lee and D.K. Oh: Char- L-rhamnose isomerase. J. Biosci. Bioeng., 85, 539―541 (1998). acterization of an Agrobacterium tumefaciens D-psicose 3- 44) B.T. Menavuvu, W. Poonperm, K. Leang, N. Noguchi et al .: epimerase that converts D-fructose to D-psicose. Appl. Environ. Efficient biosynthesis of D-allose from D-psicose by cross- Microbiol ., 72, 981―985 (2006). linked recombinant L-rhamnose isomerase: separation of prod- 23) K. Kim, H.J. Kim, D.K. Oh, S.S. Cha and S. Rhee: Crystal uct by ethanol crystallization. J. Biosci. Bioeng., 101, 340―345 structure of D-psicose 3-epimerase from Agrobacterium tume- (2006). faciens and its complex with true substrate D-fructose: a piv- 45) G.V. Levin: Tagatose, the new GRAS sweetener and health otal role of metal in catalysis, an active site for the non- product. J. Med. Food , 5,23―36 (2002). phosphorylated substrate, and its conformational changes. J. 46) Y. Lu, G.W. Levin and T.W. Donner: Tagarose, a new antidia- Mol. Biol ., 361, 920―931 (2006). betic and obesity control drug. Diabetes Obes. Metab., 10, 24) C. Ziemer and G. Gibson: An overview of prebiotics, prebiot- 109―134 (2008). ics and synbiotics in the functional food concept: perspectives 47) Y. Ishida, T. Kamiya and K. Izumori: Production of D-tagatose and future strategies. Int. Dairy J ., 8, 473―479 (1998). 3-epimerase of Pseudomonas cichorii ST-24 using recombi- 25) T. Nakakuki: Present status and future prospects of functional nant Escherichia coli. J. Ferment. Bioeng., 84, 348―350 development in Japan. J. Appl. Glycosci., 52, (1997). 267―271 (2005). 48) D.K. Oh: Tagatose: properties, applications, and biotechnologi- 26) R. Schauer: Sialic Acids―Chemistry, Metabolism, and Func- cal processes. Appl. Microbiol. Biotechnol ., 76,1―8 (2007). tion,Spriger-Verlag, New York, pp. 263―305 (1982). 49) M. Sedlak and N.W.Y. Ho: Production of ethanol from cellu- 27) S. Yamaguchi, J. Ohnishi, I. Maru and Y. Ohta: Simple and losic biomass hydrolysates using genetically engineered Sac- large-scale production of N-acetylneuraminic acid and N-acetyl charomyces yeast capable of cofermenting glucose and xylose. -D-mannosamine. Trends Glycosci. Glycotechnol ., 18, 245―252 Appl. Biochem. Biotechnol ., 113―116, 404―416 (2004). (2006). 50) M. Kuyper, M.J. Toirkens, J.A. Diderich, A.A. Winker et al .: 28) A. Datta: Regulatory role of adenosine triphosphate on hog Evolutionary engineering of mixed-sugar utilization by a kidney N-acetyl-D-glucosamine 2-epimerase. Biochemistry, 9, xylose-fermenting Saccharomyces cerevisiae strain. FEMS 3663―3770 (1970). Yeast Res., 5, 925―934 (2005). 29) T.R. Cataldi, M. Angelloti and S.A. Bufo: Method develop- 51) H.W. Wisselink, M.J. Toirkens, M.D.R.F. Berriel, A.A. Win- ment for the quantitative determination of lactulose in heat- kler et al .: Engineering of Saccharomyces cerevisiae for effi- treated milks by HPAEC with pulsed amperometric detection. cient anaerobic alcoholic fermentation of L-arabinose. Appl. Anal. Chem., 71, 4919―4925 (1999). Environ. Microbiol ., 73, 4881―4891 (2007). 30) F.J. Moreno, M. Villamiel and A. Olano: Effect of high pres- 52) H.W. Wisselink, M.J. Toirkens, Q. Wu, J.T. Pronk et al .: sure on isomerization and degradation of lactose in alkaline Novel evolutionary engineering approach for accelerated utili- media. J. Agric. Food Chem., 51, 1894―1896 (2003). zation of glucose, xylose, and arabinose mixtures by engi- 31) I. Martinez-Castro, A. Olano and N. Corzo: Modifications and neered Saccharomyces cerevisiae strains. Appl. Environ. Mi- interactions of lactose with mineral components of milk during crobiol ., 75, 907―914 (2009). heating processes. Food Chem., 21, 211―221 (1986). 32) M. Nishimukai, J. Watanabe, H. Taguchi, T. Senoura et al .: Effects of epilactose on calcium adsorption and serum lipid metabolism in rats. J. Agric. Food Chem., 56, 10340―10345 Carbohydrate Epimeraseの触媒作用，構造，その応用例 (2008). 1 33) J. Watanabe, M. Nishimukai, H. Taguchi, T. Senoura et al .: 伊藤 進 Prebiotic properties of epilactose. J. Dairy Sci., 91, 4518―4526 1 琉球大学農学部亜熱帯生物資源科学科 (2008). 34) M. Miyamoto and K. Ajisaka: Regioselectivity in β- 発酵・生命科学分野 galactosidase-catalyzed transglycosylation for the enzymatic (903―0213 沖縄県西原町千原 1 番地) assembly of D-galactosyl-D-mannose. Biosci. Biotechnol. Bio- chem., 68, 2086―2090 (2004). 現在まで 20 を超える carbohydrate epimerase が報告され 35) H. Oshima, I. Kimura and K. Izumori: Psicose contents in ており，それぞれ異なる酵素が基質の C1，C2，C3，C4， various food products and its origin. Food Sci. Technol. Res., 12, 137―143 (2006). C5，または C6 位を認識してエピマー化する．これらの 36) Y. Sun, S. Hayakawa, M. Ogawa, K. Fukada and K. Izumori: 酵素群には，たとえばaldose 1-epimerase, N-acetyl-D- Influence of a rare sugar, D-psicose, on the physicochemical and functional properties of an aerated food system containing glucosamine 2-epimerase , UDP-N-acetylglucosamine 2- egg albumin. J. Agric. Food Chem., 56, 4789―4796 (2008). 37) T. Matsuo, Y. Shirai and K. Izumori: Effects of dietary D- epimerase, cellobiose 2-epimerase, D-ribulose-5-phosphate 3- psicose on diurnal variation in plasma glucose and con- epimerase, L-ribulose-5-phosphate 4-epimerase, UDP-galactose centrations of rats. Asia Pacific J. Clin. Nutr., 10, 233―237 (2001). 4-epimerase, dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase, 38) K. Murao, X. Yu, W.M. Cao, H. Umachi et al .: D-Psicose in- GDP-mannose 3,5-epimerase, ADP-L-glycero-D-mannoheptose hibits the expression of MCP-1 induced by high-glucose stimu- lation in HUVECs. Life Sci., 81, 592―599 (2007). 6-epimerase などが含まれ， 個々の酵素の生物学的な特性， 39) M. Sato, H. Kurose, T. Yamasaki and K. Izumori: Potential 3 anthemintic: D-psicose inhibits motility, growth and productive 反応機構，タンパクの 次構造が著しく異なっている． maturity of L1 larvae of Caenorhabditis elegans. J. Nat. Med ., 本総説は，詳細が明らかになった微生物由来 carbohydrate 62, 244―246 (2008). 40) A. Murata, K. Sekita, Y. Watanabe, F. Yamaguchi et al .: A epimerase の触媒作用，構造と応用例に関する最近のト novel inhibitory effect of D-allose on production of reactive ピックスを概説したものである．

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