Kiyotaka Fujita

The difructose dianhydride I synthase/hydrolase catalyzes the reversible transformation of inulobiose into difructose dianhydride I, a component of caramel. The proposed reaction mechanism of such a transformation consists of a glycosylation step followed by a cyclization to yield the dianhydride from the disaccharide. Here, we make use of extensive hybrid QM/MM metadynamics studies to shed light on the molecular basis of it. Our simulations show that the global reaction for the transformation of inulobiose by is slightly exergonic in good agreement with the experimental data. Furthermore, we observe that the glycosylation step is the rate‐limiting step. Interestingly, our work shows that the –1 sugar of inulobiose changes from an initial E conformation into a E puckering via the conformational pathway E E / T E E . Our calculations highlight that in the cyclization step, the three residues E85, K147, and N226 are essential for the rotation of the +1 sugar in the substrate to facilitate the intramolecular attack of oxygen O1' to the anomeric carbon. Prompted by these results, we expressed and assayed the N226A variant. Enzyme activity data confirm an important role for N226 during the cyclization step but show that it is dispensable for the hydrolysis step.

The difructose dianhydride I synthase/hydrolase αFFase1 catalyzes the reversible transformation of inulobiose into difructose dianhydride I, a compound present in differ ent caramel compositions. The proposed reaction mechanism of such a transformation consists of a glycosylation step followed by a cyclization to yield the dianhydride from the disaccharide. In response to the absence of mechanistical details for the reaction, we made use of extensive hybrid QM/MM metadynamics studies to shed light on the molecular basis of it. Our simulations show that the global reaction for the transfor mation of inulobiose by αFFase1 is slightly exothermic (∆G0 =–1.3 kcal mol−1) in good agreement with the experimental data. Furthermore, we observe that the glyco sylation step is the rate-limiting step of the reaction with an energy barrier of 12.3 kcal mol−1 compared to 8.7 kcal mol-−1 for the cyclization step. Interestingly, our work shows that the–1 sugar of inulobiose changes from an initial E5 conformation into a 4E puckering via the conformational pathway E5 → E5/4T5 → E3 → 4E. Our calculations highlight that in the cyclization step, the three residues E85, K147, and N226 are es sential for the rotation of the +1 sugar in the substrate to facilitate the intramolecular attack of oxygen O1’ to the anomeric carbon. Prompted by these results, we expressed and assayed the N226A αFFase1 variant. Kinetic data confirm an important role for N226 during the cyclization step but are irrelevant for the hydrolysis step.

カラメル化糖とは，フルクトース（Fru）の加熱に伴う脱水縮合により生じる五員環構造のfructofuranose（Fruf）や六員環構造のfructopyranose（Frup）をもつ二糖及び，さらに脱水縮合して生じるdi-fructose dianhydride（DFA）やdi-heterolevulosan（DHL）等のジアンヒドロ糖である．植物の貯蔵多糖であるイヌリンやレバンがβ結合のFrufに限定されるのに対し，カラメル化糖はFrufとFrupがαもしくはβ結合にて様々な結合位置で重合した多様な異性体の混合物である．我々は，主要なカラメル化糖であるDFA I（α-Fruf-1,2′:2,1′-β-Fruf）及び，DFA III（α-Fruf-1,2′:2,3′-β-Fruf）の腸内細菌が持つ分解酵素の同定と分解代謝経路の解明に成功した．本総説では，ビフィズス菌由来の糖質加水分解酵素（GH）ファミリー172（GH172）に分類されるDFA I分解酵素と，Blautia属細菌由来のGH91 DFA III分解酵素の機能解析から見えてきた腸内細菌によるカラメル化糖の資化戦略を紹介する．

Di-fructofuranose 1,2′:2,3′ dianhydride (DFA-III) is a cyclic fructo-disaccharide, which is produced by the condensation of two fructose molecules via the caramelization or enzymatic reaction of inulin fructotransferase. A strain of Blautia producta was known to utilize DFA-III as a carbohydrate source; however, the mechanisms remain unclear. In this study, we characterized the glycoside hydrolase (GH) family 91 DFA-III hydrolase (DFA-IIIase) from B. parvula NBRC 113351. Recombinant BpDFA-IIIase catalyzed the reversible conversion of DFA-III to inulobiose, which is further degraded to fructose by the cooperative action of DFA-IIIase and GH32 β-d-fructofuranosidase. DFA-III was utilized in several Blautia species with a gene cluster for DFA-III degradation (e.g., B. parvula NBRC 113351, B. hydrogenotrophica JCM 14656, and B. wexlerae JCM 35486), but not by B. wexlerae JCM 31267, which does not possess the gene cluster. Furthermore, B. hansenii JCM 14655, which cannot metabolize fructose, could not utilize DFA-III; however, it could degrade DFA-III to fructose in the presence of DFA-III-degrading enzymes. Fecal fermentation tests showed that Blautia species are important gut microbe for degrading DFA-III. Key points: • BpDFA-IIIase is the first characterized DFA-IIIase in intestinal non-pathogenic bacteria. • DFA-IIIase is widely conserved in Blautia species. • DFA-III is degraded to d -fructose through inulobiose by the cooperative action of DFA-IIIase and β- d -fructofuranosidase.

L-Arabinofuranosides with β-linkages are present in several plant molecules, such as arabinogalactan proteins (AGPs), extensin, arabinan, and rhamnogalacturonan-II. We previously characterized a β-l-arabinofuranosidase from Bifidobacterium longum subsp. longum JCM 1217, Bll1HypBA1, which was found to belong to the glycoside hydrolase (GH) family 127. This strain encodes two GH127 genes and two GH146 genes. In the present study, we characterized a GH146 β-l-arabinofuranosidase, Bll3HypBA1 (BLLJ_1848), which was found to constitute a gene cluster with AGP-degrading enzymes. This recombinant enzyme degraded AGPs and arabinan, which contain Araf-β1,3-Araf structures. In addition, the recombinant enzyme hydrolyzed oligosaccharides containing Araf-β1,3-Araf structures but not those containing Araf-β1,2-Araf and Araf-β1,5-Araf structures. The crystal structures of Bll3HypBA1 were determined at resolutions up to 1.7 Å. The monomeric structure of Bll3HypBA1 comprised a catalytic (α/α)₆ barrel and two β-sandwich domains. A hairpin structure with two β-strands was observed in Bll3HypBA1, to extend from a β-sandwich domain and partially cover the active site. The active site contains a Zn²⁺ ion coordinated by Cys₃-Glu and exhibits structural conservation of the GH127 cysteine glycosidase Bll1HypBA1. This is the first study to report on a β1,3-specific β-l-arabinofuranosidase. Key points: • β1,3- l -Arabinofuranose residues are present in arabinogalactan proteins and arabinans as a terminal sugar. • β- l -Arabinofuranosidases are widely present in intestinal bacteria. • Bll3HypBA1 is the first enzyme characterized as a β1,3-linkage-specific β- l -arabinofuranosidase.

The cell walls of pathogenic and acidophilic bacteria, such as Mycobacterium tuberculosis and Mycobacterium leprae, contain lipoarabinomannan and arabinogalactan. These components are composed of d-arabinose, the enantiomer of the typical l-arabinose found in plants. The unique glycan structures of mycobacteria contribute to their ability to evade mammalian immune responses. In this study, we identified four enzymes (two GH183 endo-d-arabinanases, GH172 exo-α-d-arabinofuranosidase, and GH116 exo-β-d-arabinofuranosidase) from Microbacterium arabinogalactanolyticum. These enzymes completely degraded the complex d-arabinan core structure of lipoarabinomannan and arabinogalactan in a concerted manner. Furthermore, through biochemical characterization using synthetic substrates and X-ray crystallography, we elucidated the mechanisms of substrate recognition and anomer-retaining hydrolysis for the α- and β-d-arabinofuranosidic bonds in both endo- and exo-mode reactions. The discovery of these d-arabinan-degrading enzymes, along with the understanding of their structural basis for substrate specificity, provides valuable resources for investigating the intricate glycan architecture of mycobacterial cell wall polysaccharides and their contribution to pathogenicity.

D-Arabinofuranosidases (Arafases) degrade the arabinan in the cell wall of acid-fast bacteria like Mycobacterium. Synthetic arabinan fragment probes could be used to investigate the function of d-Arafases, whose synthetic studies had been reported previously. The homologue of one of the arabinan-degrading enzymes, exo-α-d-Arafase, was identified as BBDE_2040 from Bifidobacterium dentium. BBDE_2040, which is a homologue of α-d-Arafase, was also observed to hydrolyze α-d-fructofuranosides (Fruf), whose linkage is found in food. In this paper, we present synthetic studies and structural analysis of the α-d-Araf and α-d-Fruf derivatives as substrates and products of the enzymatic reaction for a thorough examination of BBDE_2040. The results indicated that BBDE_2040 is a glycoside hydrolase (GH) family 172 difructose dianhydride I synthase/hydrolase and an anomer-retaining GH.