Evaluation of the Relative Available Energy of Cyclic Nigerosylnigerose (CNN) using breath hydrogen excretion in healthy humans

Cyclic nigerosylnigerose (CNN) is a cyclic tetrasaccharide with properties distinct from those of other conventional cyclodextrins. We investigated the relative available energy of CNN in healthy humans. CNN digestibility was determined using brush border membrane vesicles from the small intestines of rats. CNN was not hydrolyzed by rat intestinal enzymes. To investigate breath hydrogen excretion, thirteen human subjects were included in a double-blind cross-over, randomized, placebo-controlled study. The effects of CNN on hydrogen excretion were compared with those of a typical nondigestible, fermentable fructooligosaccharide (FOS). In the study participants, hydrogen excretion hardly increased upon CNN and was remarkably lower than for FOS. The available energy value was determined using the fermentability based on breath hydrogen excretion and was evaluated as 0 kcal/g for CNN. CNN was hardly metabolized and hence may be used as a low-energy dietary fiber.

Structural features of a bacterial cyclic α-maltosyl-(1→6)-maltose (CMM) hydrolase critical for CMM recognition and hydrolysis

Cyclic α-maltosyl-(1→6)-maltose (CMM, cyclo-{→6)-α-d-Glcp-(1→4)-α-d-Glcp-(1→6)-α-d-Glcp-(1→4)-α-d-Glcp-(1→})is a cyclic glucotetrasaccharide with alternating α-1,4 and α-1,6 linkages. CMM is composed of two maltose units and is one of the smallest cyclic glucooligosaccharides. Although CMM is resistant to usual amylases, it is efficiently hydrolyzed by CMM hydrolase (CMMase), belonging to subfamily 20 of glycoside hydrolase family 13 (GH13_20). Here, we determined the ligand-free crystal structure of CMMase from the soil-associated bacterium Arthrobacter globiformis and its structures in complex with maltose, panose, and CMM to elucidate the structural basis of substrate recognition by CMMase. The structures disclosed that although the monomer structure consists of three domains commonly adopted by GH13 and other α-amylase–related enzymes, CMMase forms a unique wing-like dimer structure. The complex structure with CMM revealed four specific subsites, namely −3′, −2, −1, and +1′. We also observed that the bound CMM molecule adopts a low-energy conformer compared with the X-ray structure of a single CMM crystal, also determined here. Comparison of the CMMase active site with those in other enzymes of the GH13_20 family revealed that three regions forming the wall of the cleft, denoted PYF (Pro-203/Tyr-204/Phe-205), CS (Cys-163/Ser-164), and Y (Tyr-168), are present only in CMMase and are involved in CMM recognition. Combinations of multiple substitutions in these regions markedly decreased the activity toward CMM, indicating that the specificity for this cyclic tetrasaccharide is supported by the entire shape of the pocket. In summary, our work uncovers the mechanistic basis for the highly specific interactions of CMMase with its substrate CMM.

Purification, Characterization, and Gene Cloning of a Novel Maltosyltransferase from an Arthrobacter globiformis Strain That Produces an Alternating α-1,4- and α-1,6-Cyclic Tetrasaccharide from Starch

ABSTRACT A glycosyltransferase, involved in the synthesis of cyclic maltosylmaltose [CMM; cyclo-{→6)-α-d-Glcp(1→4)-α-d-Glcp(1→6)-α-d-Glcp(1→4)-α-d-Glcp(1→}] from starch, was purified to homogeneity from the culture supernatant of Arthrobacter globiformis M6. The CMM-forming enzyme had a molecular mass of 71.7 kDa and a pI of 3.6. The enzyme was most active at pH 6.0 and 50°C and was stable from pH 5.0 to 9.0 and up to 30°C. The addition of 1 mM Ca2+ enhanced the thermal stability of the enzyme up to 45°C. The enzyme acted on maltooligosaccharides that have degrees of polymerization of ≥3, amylose, and soluble starch to produce CMM but failed to act on cyclomaltodextrins, pullulan, and dextran. The mechanism for the synthesis of CMM from maltotetraose was determined as follows: (i) maltotetraose + maltotetraose → 64-O-α-maltosyl-maltotetraose + maltose and (ii) 64-O-α-maltosyl-maltotetraose → CMM + maltose. Thus, the CMM-forming enzyme was found to be a novel maltosyltransferase (6MT) catalyzing both intermolecular and intramolecular α-1,6-maltosyl transfer reactions. The gene for 6MT, designated cmmA, was isolated from a genomic library of A. globiformis M6. The cmmA gene consisted of 1,872 bp encoding a signal peptide of 40 amino acids and a mature protein of 583 amino acids with a calculated molecular mass of 64,637. The deduced amino acid sequence showed similarities to α-amylase and cyclomaltodextrin glucanotransferase. The four conserved regions common in the α-amylase family enzymes were also found in 6MT, indicating that 6MT should be assigned to this family.