In Vivo Metabolism of 1,5-Anhydro-d-fructose to 1,5-Anhydro-d-glucitol.

BACKGROUND/AIM: 1,5-Anhydro-d-fructose (1,5-AF, saccharide) and 1,5-anhydro-d-glucitol (1,5-AG) converted from 1,5-AF via the glycemic pathway have health benefits. However, this metabolism has not been sufficiently elucidated. To clarify the in vivo metabolism of 1,5-AF to 1,5-AG, porcine (blood kinetics) and human (urinary excretion) studies were conducted. MATERIALS AND METHODS: Microminipigs were administrated 1,5-AF orally or intravenously. Blood samples were obtained to analyse the kinetics of 1,5-AF and 1,5-AG. Urine samples were collected from human subjects who had orally ingested 1,5-AF, and the amounts of 1,5-AF and 1,5-AG excreted in the urine were analysed. RESULTS: In blood kinetics analysis, the time to the maximum concentration of 1,5-AF after intravenous administration was 0.5 h, whereas 1,5-AF was not observed after oral administration. The times to the maximum concentration of 1,5-AG after intravenous and oral administration were 1.5 h and 2 h, respectively. In urinary excretion, the concentration of 1,5-AG in urine rapidly increased after the administration of 1,5-AF, peaked at 2 h, whereas 1,5-AF was not detected. CONCLUSION: 1,5-AF was rapidly metabolized to 1.5-AG in vivo in swine and human.

Characterization of an inducible phenylserine aldolase from Pseudomonas putida 24-1.

An inducible phenylserine aldolase (L-threo-3-phenylserine benzaldehyde-lyase, EC 4.1.2.26), which catalyzes the cleavage of L-3-phenylserine to yield benzaldehyde and glycine, was purified to homogeneity from a crude extract of Pseudomonas putida 24-1 isolated from soil. The enzyme was a hexamer with the apparent subunit molecular mass of 38 kDa and contained 0.7 mol of pyridoxal 5' phosphate per mol of the subunit. The enzyme exhibited absorption maxima at 280 and 420 nm. The maximal activity was obtained at about pH 8.5. The enzyme acted on L-threo-3-phenylserine (Km, 1.3 mM), l-erythro-3-phenylserine (Km, 4.6 mM), l-threonine (Km, 29 mM), and L-allo-threonine (Km, 22 mM). In the reverse reaction, threo- and erythro- forms of L-3-phenylserine were produced from benzaldehyde and glycine. The optimum pH for the reverse reaction was 7.5. The structural gene coding for the phenylserine aldolase from Pseudomonas putida 24-1 was cloned and overexpressed in Escherichia coli cells. The nucleotide sequence of the phenylserine aldolase gene encoded a peptide containing 357 amino acids with a calculated molecular mass of 37.4 kDa. The recombinant enzyme was purified and characterized. Site-directed mutagenesis experiments showed that replacement of K213 with Q resulted in a loss of the enzyme activity, with a disappearance of the absorption maximum at 420 nm. Thus, K213 of the enzyme probably functions as an essential catalytic residue, forming a Schiff base with pyridoxal 5'-phosphate.