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• W2091228702 abstract "RaCE and X-ray crystallography (View interaction) Enzymatic epimerization is an important modification for carbohydrates to acquire diverse functions attributable to their stereoisomers. Epimerases and racemases are isomerases that catalyze inversion of the configuration around an asymmetric center of substrates. Changing the stereochemistry of the hydroxyl substituent has biological significance in all branches of life. In humans, mutations found in the UDP-glucose epimerase gene would be lethal [1]. In plants, d-ribulose-5-phosphate 3-epimerase is a key enzyme in the Calvin cycle and the oxidative pentose phosphate pathway [2, 3]. In pathogenic bacteria, epimerases are involved in the production of complex carbohydrate polymers used in their cell walls and envelopes to protect them from the host immune system [4]. Cellobiose 2-epimerase (CE) (EC 5.1.3.11), which was first found in the culture fluid of an anaerobic ruminal bacterium, Ruminococcus albus [5], catalyzes reversible epimerization of the d-glucose residue at the reducing end of oligosaccharides linked by β-1,4-glycosidic linkages, such as cellobiose, lactose, and 4-O-β-d-mannosyl-d-glucose to a d-mannose residue. Although this is the sole enzyme responsible for catalyzing epimerization of the 2′-OH group of non-modified oligosaccharides, it has been shown to be widely distributed not only in anaerobes [6-8] but also in aerobes [9, 10]. In R. albus and Bacteroides fragilis, the CE gene comprises an operon together with the β-1,4-mannanase gene and the 4-O-β-mannosylglucose phosphorylase (EC 2.4.1.281, MP) gene encoding the enzyme catalyzing specific phosphorolysis of 4-O-β-d-mannosyl-d-glucose to α-d-mannose 1-phosphate and d-glucose [11, 12]. Thus, CE may have a role in the metabolism of mannan; i.e., mannobiose produced by hydrolysis of mannan with β-1,4-mannanase is epimerized to 4-O-β-d-mannosyl-d-glucose by CE, and then the epimerized product is phosphorylated by the phosphorylase [11, 12]. CE is an attractive enzyme to produce epilactose (4-O-β-d-galactosyl-d-mannose) from lactose. Epilactose is a non-digestible oligosaccharide, and enhances proliferation of bifidobacilli and lactobacilli in the gut [13]. This stimulation of growth of beneficial bacteria suppresses the conversion of primary bile acid to secondary bile acid, which is considered a risk factor for colon cancer, and enhances absorption of minerals, including calcium, magnesium, and zinc [13, 14]. Furthermore, epilactose increases intestinal absorption of calcium through the paracellular route [15]. For application of epilactose as a functional foodstuff, practical methods for preparation of epilactose have been developed [16, 17]. CE enzymes belong to the N-acetyl-d-glucosamine 2-epimerase (AGE) superfamily. In this superfamily, the structures of two epimerases, i.e., AGE and aldose–ketose isomerase YihS have been reported, and their catalytic mechanisms have been suggested (Fig. 1 ) [18]. The three-dimensional structures of AGEs from Anabaena sp. CH1 (aAGE) (PDB ID: 2GZ6) [18], and porcine kidney (pAGE) (PDB ID: 1FP3) [19] have been determined, and the catalytic mechanism of AGE was postulated based on the results of structural and site-directed mutational studies [18]. In the case of AGE, two histidines, His239 and His372 (numbered with reference to aAGE) at the catalytic center surrounded by inner helices, were responsible for general acid/base catalysis to achieve interconversion between N-acetyl-d-glucosamine (GlcNAc) and N-acetyl-d-mannosamine (ManNAc). These two histidines interacted electrostatically with specific glutamates to stabilize the positive charge of their imidazole rings. Furthermore, the structure of YihS, which catalyzes isomerization of an unmodified sugar, has been also determined. The structures of YihS from Escherichia coli (EcYihS) (PDB ID: 2RGK) and Salmonella enterica (SeYihS) (PDB ID: 2AFA), which show high levels of activity toward mannose and glucose [20], are similar to that of AGE. Although RaCE shows sequence similarity with low identity to AGE and YihS, several amino acids in the catalytic centers of AGE and YihS are well conserved in CE (Arg52, His243, Glu246, Trp249, Trp304, Glu308, and His374 in RaCE), and mutational experiments with RaCE indicated that these residues are critical for the catalytic activity [21]. However, the substrate specificities of CE, AGE, and YihS differ from each other in terms of substrate chain length and in the chemical group at the C2 position of the substrate. While AGE reacts with modified sugars, CE and YihS react with unmodified sugars. Moreover, epimerization by AGE and YihS is specific to monosaccharides, whereas CE reacts with oligosaccharides. Although the common epimerization reaction occurring on deprotonation from a chiral carbon was proposed for both AGE and YihS, the deprotonation of unmodified sugars such as in YihS is still unclear. In this study, we determined the crystal structure of recombinant RaCE to examine the catalytic mechanism of CE for unmodified oligosaccharides. The key residues for discriminating the substrate chain length and epimerization toward unmodified sugar were suggested based on comparison of the catalytic center of RaCE with those of AGEs and YihSs. Moreover, mutational analysis reinforced the importance of the key residue for the enzymatic reaction of RaCE, and we discuss the roles of the key residues for deprotonation during epimerization. The materials and methods of expression, purification, crystallization, and structure determination are provided in the Supplementary text. The coordinates of RaCE have been deposited in the RCSB Protein Data Bank with accession code 3VW5. Mutanted CEs, in which His184 was substituted by Ala (H184A) and Asn (H184N), and Trp369 was substituted by Ala (W369A) were prepared. Site-directed mutagenesis was performed with a PrimeSTAR Mutagenesis Basal Kit (Takara Bio, Otsu, Japan) using the expression plasmid of the wild-type enzyme and appropriate specific primers (Table S2). The resulting plasmids were introduced into E. coli BL21 (DE3), and the mutant enzymes were produced and purified as described above. Enzyme activities of the mutant enzymes were measured as described previously [21]. The structure of RaCE was determined at 2.6 Å resolution by the molecular replacement method using the structure 3GT (PDB ID) as a search model. The three molecules of RaCE in the asymmetric unit were clearly built and 156 water molecules were located (Fig. 2 ). Three monomers in an asymmetric unit were related by non-crystallographic threefold symmetry, and were superposed well on each other with an average root mean square deviation (r.m.s.d.) of 0.19 Å for 370 Cα atoms (95% of the entire residues of RaCE). The overall structure of RaCE represented a typical (α/α)6 barrel fold comprised of six inner helices (α2, α4, α6, α8, α10, α12) and a further six lateral helices (α1, α3, α5, α7, α9, α11) (Fig. 2) oriented in an antiparallel manner. Although sequence similarity between CE and the other sugar epimerases AGE and YihS is low (about 15%) (Fig. 3 ), the structure of RaCE showed a considerably similar architecture to those of pAGE, aAGE, EcYihS, and SeYihS according to DALI [22] search with r.m.s.d. of 2.6, 2.5, 2.8 and 2.3 Å, and Z-scores of 42.9, 40.4, 42.9, and 42.9, respectively. Notably, sequence alignment with structural comparison showed that 11 residues were completely conserved among CEs, AGEs, and YihSs. Seven of these residues were located in the catalytic center (Fig. 4 ) and formed a hydrogen bond network (Fig. 5 ). Structural differences among CE, AGE, and YihS were observed only in two loops (β7–β8 and β11–β12 loops in CE; β5–β6 and β9–β10 loops in AGE; β9–β10 and β11–α8 loops in YihS) wrapped on one side of the barrel. In the case of SeYihS, the β11–α8 loop between β11 and α8 helix is high flexible, and it could not be built in the apo form, whereas the catalytic center of SeYihS–mannose is hindered by this loop (closed form) (Fig. 6 ). In the present structure, the putative catalytic center of RaCE was exposed to solvent (open form), and the β7–β8 loop (corresponding to β9–β10 loop in SeYihS) seemed to be flexible with high temperature factors, suggesting that this β7–β8 loop may compensate for the role of the β11–α8 loop in SeYihS as the lid of the substrate-binding site. Such differences in flexible loops were also observed in AGE; the α5–β6 loop was disordered in aAGE, and exhibited a high degree of flexibility with high temperature factors in pAGE. To discuss the substrate specificity of RaCE, a model of RaCE in complex with mannose was built by superposing the structure of SeYihS–mannose complex on that of RaCE. As in the structure of SeYihS–mannose complex, the mannose in the RaCE–mannose model was fitted into the substrate-binding cleft with nearly parallel orientation to the plane of the indole group of Trp304 (corresponding to Trp316 in SeYihS) and 6′-OH was close to the side chain of Arg52 (corresponding to Arg55 in SeYihS). These residues (Arg52 and Trp304) were also shown to be required for catalytic activity of RaCE by mutational analysis [21]. In fact, aromatic residues are generally involved in recognizing carbohydrates by stacking [23], and near-stacking interactions were reported in carbohydrate-binding proteins, such as glycoside hydrolases CelA from Clostridium thermocellum [24] and CMCax from Acetobacter xylinum [25]. Moreover, the superposed model suggested another possible stacking interaction between the substrate and Trp369, which was positioned toward the extended direction from the 4′-OH of mannose (Fig. 7 ). Considering the sequence conservation (Fig. 3) of Trp369 only in the CE family, which reacts toward a disaccharide bound second sugar moiety from the reducing end of β-1,4-linked oligosaccharide, Trp369 located at the entrance of the catalytic center in RaCE may be required to bind a disaccharide; i.e., the relative orientation of aromatic residues to the substrate may be responsible for substrate chain length discrimination. In conclusion, the absolutely conserved residues Arg52 and Trp304 in RaCE, AGE, and YihS may recognize the common parts of the substrates in three kinds of enzymes, whereas the conserved residue Trp369 in the RaCE family seems to recognize the specific parts of the substrates of CE. Such importance of Trp369 for the recognition of disaccharide was verified by mutational experiment; point mutation of Trp369 to Ala (W369A) resulted in the complete loss of the epimerization activity even at 50-fold higher concentration of the wild-type RaCE (Fig. 8 ). In addition to the putative residues for substrate specificity described above, the residues for catalysis also showed structural and sequence conservation among CE, AGE, and YihS. His239 and His372 of aAGE were considered as general acid/base catalysts due to their arrangements as well as the markedly reduced activity at acidic pH [18]. In the structure of SeYihS–mannose complex, His248 corresponding to His239 of aAGE was believed to transfer a proton during epimerization, whereas His383 corresponding to His372 of aAGE was a candidate for an acid/base catalyst as it was located within hydrogen bonding distance to 1′-OH of mannose [20]. In the case of RaCE, His243 and His374 correspond to the histidines involved in acid/base catalysis of epimerization in AGE and YihS. As shown in Fig. 9 , the side chain of His243 had a different orientation to the corresponding residues of His239 in aAGE and His248 in SeYihS, whereas the other key residue His374 was structurally conserved with His372 in aAGE and His383 in SeYihS as His374 of RaCE participated in formaton of an extensive hydrogen bond network with most conserved residues of His184, Glu187, Glu246, Trp249, Glu308, Tyr373, and Arg377 (Fig. 5). On the other hand, the interaction around His243 may be unique to CE as Asn180 and Ser240 are only conserved in the CE family but not in the other two families of epimerases. Although these catalytic histidines interacted with neighboring residues in slightly different manners among CE, AGE, and YihS, distinct enzymatic activities are exerted with a similar catalytic center, as these completely conserved histidine residues were located at the same positions in three types of sugar epimerase. Moreover, site-directed mutational studies showed that H243A and H374A of RaCE completely lost epimerization activities toward cellobiose and lactose [21], indicating the necessity of these histidines for enzymatic activity. Therefore, we propose that His374 and His243 act as a general acid/base catalyst similar to those of AGE and YihS. Interestingly, His176 in SeYihS–mannose structure was located in proximity to both 1′-OH and 2′-OH of mannose [20]. The histidine residue His184 corresponding to His176 in SeYihS was also found in RaCE. The relative position of His184 to the bound mannose in the RaCE–mannose model was similar to that of His176 in SeYihS (Fig. 7). His184 mutants, H184N and H184A, showed no enzyme activity even at 100-fold higher concentration of the wild-type enzyme (Fig. 8), suggesting that His184 of RaCE is an essential amino acid residue for the catalytic activity. When isomerization through proton abstraction occurs at the chiral center, isomerases are thought to use distinct deprotonation strategies. Some amino acid racemases utilize pyridoxal phosphate (PLP) as an electron sink for effective deprotonation on α-carbon. The π-electron conjugated system from the pyridine ring of PLP to the α-amino group of amino acid is generated through a Schiff base between PLP and the substrate amino acid, which readily causes Cα–H bond cleavage [26]. In the case of AGE, π-electron delocalization caused by the resonance structures of the N-acetyl group bound to C2 is favorable for deprotonation on C2. When isomerization occurs at a chiral center adjacent to a carbonyl group, the pK a of the hydrogen connecting the stereogenic carbon is lowered by depolarization of a functional group attached to the same carbon, accomplishing C–H bond cleavage. Then, the CC double bond abstracts a proton from an acid to remake the C–H bond. However, in the cases of CE and YihS, localization of electrons on the 2′-OH of the reducing end of the substrate makes deprotonation from the C2 position difficult. To overcome unfavorable deprotonation of the α-proton in RaCE, His184, which is also located on the surface of the substrate-binding pocket, may help to depolarize 2′-OH described above by non-covalent interaction between the amino group of the imidazole ring and the 2′-OH group of the substrate. The polar interaction of Glu187-His184 is similar to that of Glu308-His374, allowing His184 to assist in polarization of the 2′-OH group. The observation that the third histidine residue is found in both CE and YihS acting on unmodified sugars implies that the histidine residue is crucial for epimerization toward an unmodified sugar. Therefore, we propose a new reaction mechanism of CE, in which CE generally catalyzes the epimerization reaction with the three most important histidines called the “triplet histidine center” (9, 10 C double bond abstracts a proton from the acid catalyst following generation of the cis-enediol intermediate. As a result, the configuration around C2 can be changed. Further studies involving structural analysis of CE complexed with substrates will be required to confirm the properties of these residues of CE. We thank the staff of beamline BL41XU at SPring-8 (Proposal No. 2011A1062, 2011B1385) for assistance with data collection. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2013.02.007. Supplementary data Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article." @default.
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• W2091228702 title "Crystal structure of <i>Ruminococcus albus</i> cellobiose 2-epimerase: Structural insights into epimerization of unmodified sugar" @default.
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