Synthesis and structural analysis of d-fructofuranosylated compounds for the analysis of GH172 difructose dianhydride I synthase/hydrolase

Introduction

For the functional analysis of d-arabinofuranosidases (Arafases) that degrade the cell wall arabinan from acid-fast bacilli, such as Mycobacteria [1], [2], [3], some synthetic arabinan fragment probes whose synthesis had been reported previously [4] could be used. As part of a series of new enzyme studies, the homologue of one of the arabinan-degrading enzymes, exo-α-d-Arafase [5], was identified as the BBDE_2040 gene product from Bifidobacterium dentium JCM 1195 (Fig. 1a) [6]. Immediately after the observation, its X-ray structure was shown [7, 8]. The crystal structure of BBDE_2040 was determined at 1.96 Å resolution as a ligand free structure (PDB ID: 7V1V). The asymmetric unit contained a hexamer with D ₃ dihedral symmetry. Each monomer of the asymmetric unit consisted of three distinct secondary structures: unique β-jelly roll folds 1 (residues 3–200, dotted frame) and 2 (201–396, solid frame), and α-helix with 52 amino-acid residues at the C-terminus (397–448, long dotted frame) (Fig. 1b). BBDE_2040 belongs to the DUF2961 (PF11175) family in the Pfam database [9], and although no functional information is available, the crystal structure of a hypothetical protein, BACUNI_00161, from Bacteroides uniformis ATCC 8492 (PDB ID: 4KQ7) is available in the PDB from a structural genomics project. The amino acid sequence of BBDE_2040 also showed no significant similarity to any known enzymes. The new glycoside hydrolase family (GH172) has been established by the unique characterization of BBDE_2040.

Fig. 1:

BBDE_2040 (GH172 DFA I synthase/hydrolase). DFA: difructose dianhydride. (a) Organization of BBDE_2040 and surrounding genes in the genome of B. dentium JCM 1195. The numbers correspond to the locus tag (BBDE_XXXX). (b) X-ray structure of BBDE_2040 (PDB ID: 7V1X). β-Jelly roll 1, and 2 and C-terminal-helix are indicated as dotted, solid, and long dotted circles, respectively. A calcium ion (black sphere) and β-d-Fruf (gray stick) are shown in the active site (solid box). The domain architecture is shown on the right. C_ter: C-terminus; N_ter: N-terminus; DUF: domain of unknown function (adapted from Fig. 2 in Ref. [7]). (c) BBDE_2040 catalyzes both dehydration of inulobiose and hydrolysis of DFA I through formation and hydrolysis of α-fructofuranoside, respectively.

The enzymatic activity of BBDE_2040 was found to resemble that of α-d-Arafase as a homologue; it also hydrolyzes α-d-fructofuranosides (Fruf) [5], whose linkage is found in sucrose caramel and fermented plant extracts [10, 11]. The active site of BBDE_2040 is at the interface of the two protomers found in the structures of BBDE_2040 complexed with d-fructose (PDB ID: 7V1X) [7] (Fig. 1b) and d-arabinose (PDB ID: 7V1W) [7] at a resolution of 1.76 and 1.87 Å, respectively. Among the important amino acid residues surrounding the sugar ligands in the active site, two acidic residues, Glu291 and Glu270, are located close to the anomeric C2 atom in the β- and α-faces and are suggested to be nucleophile and acid/base catalyst residues, respectively [7]. A metal ion was observed close to the active site and is suggested to be Ca²⁺, which formed a hydrogen bond with the C4 hydroxy group of Fruf through one of the two coordinating water molecules [7].

For a detailed analysis of the activity of BBDE_2040, α-d-fructo/α-d-arabinofuranosidase (αFFase1), the substrate probes, such as p-nitrophenyl (pNP) α-d-Fruf 3 and α-d-Araf 4, were synthesized through the neighboring group participation method [12] for 1,2-trans glycosylation, and their properties were studied, such as the stability and activity as the substrate of BBDE_2040. We also report a study that identified the substrate and the mechanism of catalysis of BBDE_2040. Later it was found that BBDE_2040 is also difructose dianhydride I synthase/hydrolase [7] (Fig. 1c). The substrates of enzyme were fructobioses such as β-d-Frup-(2→1)-d-Fru observed in caramelized fructose and inulobiose (β-d-Fruf-(2→1)-d-Fru), and the products are unique α-linked dianhydrides such as diheterolevulosan II (DHL II, α-d-Fruf-1,2ʹ:2,1ʹ-β-d-Frup) and difructose dianhydride I (DFA I, α-d-Fruf-1,2ʹ:2,1ʹ-β-d-Fruf) [13], respectively.

Synthesis and structural analysis of d-fructofuranosylated probe as the substrate for glycoside hydrolase from B. dentium BBDE_2040

Although we found the new enzyme BBDE_2040 as a bifidobacterial homologue of α-d-Arafase, which may hydrolyze the non-reducing terminal α-d-Araf linkage [3], we focused on the d-Fruf found in the plant polysaccharides because its monosaccharide residue of fructans such as inulin and levan are probably present in living conditions for B. dentium. Because the structure of d-Fruf is identical to that of 1-hydroxymethyl-substituted d-Araf (Fig. 2a, b), we hypothesized that the bifidobacterial homologue could hydrolyze both α-d-Fruf and α-d-Araf glycosides. However, natural plant fructans are known to consist of β-d-Fruf as β-d-Fruf-(2→1)- and β-d-Fruf-(2→6)-repeating units of inulin and levan, respectively. The natural linkage appeared to be opposite to the expected substrate, α-d-Fruf for BBDE_2040. Interestingly, α-d-Fruf glycoside was found to be α-d-Fruf-(2→6)-d-Glc, which was synthesized from d-Fru and d-Glc at 140 °C for 45 min. α-d-Fruf-(2→6)-d-Glc has been used by the B. longum, B. breve and Lactobacillus [11]. This heating process of sugar molecules has been part of our daily cooking since human began using fire for it. We also discovered that the newly discovered enzyme, α-d-fructofuranosidase (αFFase1), hydrolyzed methyl α-d-Fruf 1 and methyl α-d-Araf 2 [14, 15] (Fig. 2d, lanes 3–6).

Fig. 2:

BBDE_2040 (αFFase1) has α-d-fructofuranosidase and α-d-arabinofuranosidase activities. (a, b) Structural similarity between fructofuranoside (a) and arabinofuranoside (b). (c) Substrates 1–4 for Bifidobacterium dentium BBDE_2040. (d) Activity of BBDE_2040 toward substrates 1–3. d-Fructose (Fru₁) and fructobiose (Fru₂, d-Frup-β-(2→1)-d-Fru) were used as the standard. Substrates 1–3 were incubated in Na acetate (pH 6.0) buffer with/without purified BBDE_2040 for 24 h at 37 °C. (e) Hydrolysis of substrates 3 and 4 under various conditions. d-Fructose (Fru₁) was used as the standard. *: 3 was used at 10 times higher concentration. ^†: TLC was carried out immediately after melting the frozen mixture. (f) Hydrolysis of pNP α-d-Fruf 3 at 37 or 25 °C. The conversions were calculated by ImageJ using TLC [16, 17]. The figures (e and f) are adapted from Fig. S1 (b and a) in Ref. [7], respectively.

For a more detailed analysis of the bifidobacterial homologue enzyme, the substrate probe (p-nitrophenyl (pNP) α-d-Fruf 3 [7]) was prepared according to previously reported method [18], [19], [20] through neighboring group participation method [12]. As shown in Scheme 1, pNP α-d-Fruf 3 was synthesized using a procedure similar to that used for pNP α-d-Araf 4 [18]. Under the equilibrium of d-fructose between pyranose and furanose instead of d-arabinose, treatment with tert-butyldiphenylsilyl (TBDPS) chloride was carried out to afford 1,6-di-O-TBDPS-protected d-Fruf, which was then acetylated to give the triacetate form. The resulting glycosyl acetate was treated with p-toluenethiol in the presence of BF₃·OEt₂ to afford α:β 2.0:1 mixture of p-tolyl (Tol) 3,4-di-O-acetyl-1,6-di-O-TBDPS-d-Fruf in good yield. The mixture was then converted to p-Tol 1,3,4,6-tetra-O-acetyl-d-Fruf in two steps. The introduction of pNP glycosides was performed with p-nitrophenol under N-iodosuccinimide (NIS)–silver trifluoromethanesulfonate (AgOTf) conditions [12]. The reaction was carried out at −20 °C to give pNP 1,3,4,6-tetra-O-acetyl-α-d-Fruf selectively through neighboring group participation kinetically from 3-O-Ac, as shown in Scheme 1. A smaller ³ J _H3–H4 value for α-Fruf (∼3.5 Hz) [10] than that of β-Fruf (∼8.0 Hz) [22] and an observed Nuclear Overhauser Effect (NOE) Difference Spectroscopy between C3-H of α-Fruf and o-H of p-nitrophenyl group confirmed the structure of the pNP 1,3,4,6-tetra-O-acetyl-α-d-Fruf. Subsequent deprotection of the acetyl groups of pNP 1,3,4,6-tetra-O-acetyl-α-d-Fruf at 0 °C afforded the desired compound 3, which was subjected to the homologue of α-d-Arafase.

Scheme 1:

Synthesis of pNP α-d-Fruf 3. The detailed procedures were shown in Supplementary Material.

d-Arabinofuranosylated probe as the substrate and inhibitor for DFA I synthase/hydrolase

The hydrolysis of pNP α-d-Fruf substrate probe 3 with BBDE_2040 was then tested. These results indicated that pNP α-d-Fruf 3 was hydrolyzed to d-Fru even in the absence of BBDE_2040 at 37 °C (Fig. 2d, lanes 1–2, Fig. 2e, lanes 1–2). This could be due to the higher leaving ability of the pNP group than methyl group in the methyl α-d-Fruf 1 and possibly due to the intramolecular effect of the nucleophilic hydroxymethyl group substituted at the anomeric carbon in the Fruf structure which is not present in the pNP α-d-Araf molecule 4. Under both strongly basic (pH 12) and acidic (pH 2) conditions the hydrolysis of pNP α-d-Fruf 3 was observed in the absence of BBDE_2040 (Fig. 2e, lanes 5–6). Hydrolysis was also obvious at 100 °C within 5 min at the optimized pH for BBDE_2040 (Fig. 2e, lane 7). An improvement in the stability of pNP α-d-Fruf 3 was observed at −20 °C in the absence of BBDE_2040 (Fig. 2e, lane 8); however, hydrolysis was clearly detected when pNP α-d-Fruf 3 was used at a 10-fold concentration (Fig. 2e, lanes 9–10). Subsequently, pNP α-d-Araf 4 was also prepared as previously described for the synthesis of arabinofuranoside derivatives [4, 17] and enantiomeric l-Araf derivatives [19], [20], [21]. We then examined the hydrolysis of pNP α-d-Araf 4 with and without BBDE_2040 and confirmed the stability and turnover of pNP α-d-Araf 4 in the absence and presence of BBDE_2040, respectively (Fig. 2e, lanes 3–4).

The hydrolysis of pNP α-d-Fruf 3 with BBDE_2040 was monitored at 25 °C because it was more stable at 25 °C (Fig. 2f, lanes 4–8) than at 37 °C (Fig. 2f, lanes 1–3) in the absence of BBDE_2040. In the presence of BBDE_2040, the hydrolysis at 25 °C was promoted by the enzyme (Fig. 2f, lanes 9–12), as clearly indicated by the higher conversions at the same incubation times than that in the absence of it. The results of this experiment clearly suggested that the pNP α-d-Fruf 3 is a substrate for BBDE_2040.

pNP α-d-Araf 4 was, however, used for further analysis because pNP α-d-Fruf 3 was found to be too unstable to be used as the substrate. On the contrary, pNP α-d-Araf molecule 4 is stable and easy to use for analyzing all furanose and pyranose anomers based on the peaks of C1-H and the values of ³ J _H1–H2 in the monitoring of the reaction by NMR (Fig. 3).

Fig. 3:

Monitoring of the hydrolysis of pNP α-d-Araf (4) with BBDE_2040. (a) Scheme for the hydrolysis of 4 with BBDE_2040. (b) ¹H NMR spectra monitoring the activity toward 4 in D₂O referenced at DOH. (c) An expanded chart of the area boxed in panel A referenced at H1 of α-d-Araf. The characteristic chemical shifts and J-coupling constants at anomeric C1-H of pNP α-d-Araf, and all 4 isomers of d-Ara (β-d-Araf, α-d-Araf, α-d-Arap, and β-d-Arap) are indicated by arrows (adapted from Fig. 2 in Ref. [7]).

The enzymatic hydrolysis of pNP α-d-Araf 4 with BBDE_2040 was performed with NMR monitoring, as shown in Fig. 3a. pNP α-d-Araf 4, showing the anomeric hydrogen signal as a doublet at 6.01 ppm (³ J _H1–H2 = 1.2 Hz), was clearly disappeared after 1 min of treatment and hydrolyzed completely to d-Araf which was isomerized to reach equilibrium by mutarotation of d-Araf. The ¹H NMR spectra within anomeric region clearly showed the initial presence of α-d-Araf as the major furanoside (Fig. 3b), with the H-1 signal as a doublet at 5.39 ppm (³ J _H1–H2 = 2.8 Hz) and a much weaker signal of H-1 for β-d-Araf as a doublet at 5.45 ppm (³ J _H1–H2 = 4.4 Hz). The subsequent appearance of H-1 signals as doublets at 5.38 ppm (³ J _H1–H2 = 3.6 Hz) and 4.66 ppm (³ J _H1–H2 = 8.0 Hz) followed by increase in the intensity under the equilibrium conditions within 30 min, indicated the formation of α-d-Arap and β-d-Arap as more stable pyranose forms, respectively. In addition, hydrolysis by BBDE_2040 was inhibited in the presence of tolyl and methyl 1-thio-α-d-Araf as a competitive inhibitor by 66 and 29%, respectively, after incubation with 3-fold concentrations of inhibitors on substrate (pNP α-d-Araf 4) at 37 °C for 1 h. These findings strongly suggested that BBDE_2040 is an anomer-retaining GH.

Development of substrates for DFA I synthase/hydrolase

As reported in the previous paper [7], we prepared caramelized fructose, which was expected to contain an α-d-Fruf structure (Fig. 4) and examined the enzymatic hydrolysis of caramel with BBDE_2040. We found a substrate peak in the caramelized fructose (Fig. 4b) that was converted to a new peak after the enzymatic treatment (Fig. 4c). After isolating the substrate from the caramelized fructose by gel-filtration chromatography on a Bio-Gel P-2 column [7], the conversion was reproduced to obtain a clean peak with high conversion efficiency. As the enzyme is α-d-Frufase, a homologue of α-d-Arafase, we expected that the substrate would contain an α-d-Fruf linkage in the molecule and would be hydrolyzed after treatment. The substrate and product samples from the in vitro enzymatic assays were analyzed using an electron spray ionization time-of-flight mass spectrometer (JEOL AccuTOF JMS-T700LCK), with CF₃CO₂Na as the internal standard. For the analysis, 100% methanol was used as the solvent and the electrospray ionization source was operated in the positive ion mode. The substrate and product were detected as [M+Na]⁺ (m/z = 365 and 347, respectively) (Fig. 4g). This indicated that the product must be corresponding anhydride of the disaccharide. To confirm the structure unambiguously, the substrate and product were fully acetylated. The corresponding acetates from the substrate and product were octaacetate ([M+Na]⁺ m/z = 711) and hexaacetate ([M+Na]⁺ m/z = 599), respectively (Fig. 4h). This result also indicated that the product must be corresponding anhydride of the disaccharide. We also analyzed the product and the hexaacetate of the product by NMR analysis using 1D and 2D techniques (Fig. 4i, and j). HMBC and chemical shift change by acetylation of hydroxy groups suggested that the product contains Fruf and Frup and that the linkages between the two fructose residues are d-Fruf-(2➝1)-d-Frup and d-Frup-(2➝1)-d-Fruf. ¹³C NMR data indicated that the anomeric configurations for d-Frup (95.7 and 95.5 ppm) and d-Fruf (102.5 and 101.5 ppm) were β and α, respectively. In addition, the ³ J _H3–H4 value (2.0 Hz) of d-Fruf also indicated the α-linkage formed in this enzymatic reaction. Thus, NMR analysis of the substrate combined with MS analysis suggested that BBDE_2040 catalyzes the dehydration of fructobiose, β-d-Frup-(2➝1)-d-Fru to create an unexpected difructose dianhydride, α-d-Fruf-1,2ʹ:2,1ʹ-β-d-Frup (diheterolevulosan II, DHL II) (Fig. 4g).

Fig. 4:

Treatment of caramelized fructose with Bifidobacterium dentium BBDE_2040. (a–c) HPLC profile of treatment of caramelized fructose. (d–f) HPLC profile of treatment of purified unknown substrate obtained from caramelization of fructose. (a, d) Fructose as standard. (b) Caramelized fructose. (c) After treatment of caramelized fructose with BBDE_2040 (Fig. 4b, c are adapted from Fig. 3a in Ref. [7]). (e) Purified unknown substrate. (f) After treatment of purified unknown substrate with BBDE_2040. (g) Unexpected reaction pathway for the treatment of purified substrate (m/z = 365) with BBDE_2040 to the product (m/z = 347). MALDI-TOF mass spectra were recorded on a SHIMADZU Kompact MALDI AXIMA CFR spectrometer with 2,5-dihydroxybenzoic acid as the matrix. (h) Acetylation of purified unknown substrate (e) and product (f) afforded the octaacetate (m/z = 711) and hexaacetate (m/z = 599), respectively. (I) NMR data of purified unknown substrate in D₂O (shown in Table 1 of Ref. [7]). (j) NMR data of hexaacetate of purified unknown substrate in CDCl₃ (shown in Fig. S7F of Ref. [7]). ¹H NMR and ¹³C NMR spectra were recorded on a JEOL ECX400 spectrometer. Assignments were made by standard pfg COSY, pfg HMQC or pfg HMBC, and so on.

Based on the hydrolysis retention mechanism, we observed that α-fructofuranose should be formed initially before cyclization by forming an α-fructofuranosidic linkage at the reducing fructose residue via a similar retention mechanism. However, the structure of the non-reducing residue in fructobiose seems to be compatible. Although it has a different β-d-Fruf linkage than β-d-Frup in previous β-d-Frup-(2→1)-d-Fru for DHL II, inulobiose (β-d-Fruf-(2→1)-d-Fru) [23, 24] is expected to be a favorable carbon source for bifidobacteria in the large intestine.

As shown in Fig. 5, inulobiose, the substrate of BBDE_2040, was transformed to DFA I (Fig. 5a–b, e) and DFA I, the substrate of BBDE_2040, hydrolyzed slightly to the inulobiose under equilibrium conditions (Fig. 5c–d, 5e). The reactions reached an equilibrium ratio of DFA I:inulobiose = 89.1:10.9 by HPAEC-PAD using inulobiose or DFA I as a substrate (Fig. 5e). As mentioned previously, the fructose residue at the reducing end is interconverted to α-fructofuranose before catalysis by BBDE_2040 to DFA I with the essential formation of α-fructofuranoside. This conversion is reversible and reaches equilibrium (Fig. 5f).

Fig. 5:

Interconversion between inulobiose and DFA I with BBDE_2040. (a–d) HPLC profile of the interconversion. (e) The interconversion under equilibrium conditions (adapted from Fig. 4B in Ref. [7]). (f) Chemical structures for interconversions. (g–j) NMR spectra of the interconversion (adapted from Fig. S10A in Ref. [7]). (k) ¹H and ¹³C NMR data of DFA I (shown in Fig. S10B of Ref. [7]). (l) Plausible double inverting mechanism of action for BBDE_2040. (a, g) Inulobiose; (b, h) Inulobiose treated with BBDE_2040 for 24 h; (c, i) DFA I treated with BBDE_2040 for 24 h; (d, j) DFA I.

Furthermore, the conversion between inulobiose and DFA I by BBDE_2040 was monitored by ¹H NMR (Fig. 5g, and j) in both directions. As shown in Fig. 5k, the ¹H and ¹³C NMR data of the DFA I produced from inulobiose with BBDE_2040 were identical to those previously reported [25]. The formation of α-Fruf linkage from inulobiose was unambiguously confirmed by the distinctive values of ¹ J _H3–H4 coupling constants (2.4 Hz) and ¹³C chemical shifts at C3 (82.1 ppm). These findings suggest that BBDE_2040 is a difructose dianhydride I synthase/hydrolase via a double inverting mechanism of action (Fig. 5l), that has been established as a member of novel GH172 family.

Conclusion

For a detailed analysis of BBDE_2040 with α-d-Frufase and α-d-Arafase activities, probes used as the substrates (pNP α-d-Fruf 3 and pNP α-d-Araf 4) were synthesized through the neighboring group participation method [12] for 1,2-trans glycosylation. Since the pNP α-d-Fruf 3 was found to be unstable even in the absence of degrading enzymes possibly due to the intramolecular effect of the nucleophilic hydroxymethyl group substituted at the anomeric carbon in the Fruf structure, pNP α-d-Araf 4 was efficiently used for the chemical analysis of the BBDE_2040 mechanism. NMR analysis of the enzymatic hydrolysis using 4 showed the initial formation of α-d-Araf, which strongly indicated the anomer-retaining mechanism of BBDE_2040.

Structural analysis of both substrate and product from caramelized fructose revealed that the enzyme catalyzes the production of diheterolevulosan (DHL II) through α-d-Fruf formation. We also found that inulobiose, a common fructobiose molecule in inulin degradation products, also worked as a substrate to form difructose dianhydride I (DFA I) and that this enzymatic reaction reached an equilibrium through the formation and hydrolysis of α-d-Fruf linkage in the DFA I molecule. The results indicate that BBDE_2040 is a novel GH172 family difructose dianhydride I synthase/hydrolase [26].