Molecular cloning and biochemical characterization of a novel erythrose reductase from Candida magnoliae JH110

<p>Abstract</p> <p>Background</p> <p>Erythrose reductase (ER) catalyzes the final step of erythritol production, which is reducing erythrose to erythritol using NAD(P)H as a cofactor. ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics. Although ERs were purified and characterized from microbial sources, the entire primary structure and the corresponding DNA for ER still remain unknown in most of erythritol-producing yeasts. <it>Candida magnoliae </it>JH110 isolated from honeycombs produces a significant amount of erythritol, suggesting the presence of erythrose metabolizing enzymes. Here we provide the genetic sequence and functional characteristics of a novel NADPH-dependent ER from <it>C. magnoliae </it>JH110.</p> <p>Results</p> <p>The gene encoding a novel ER was isolated from an osmophilic yeast <it>C. magnoliae </it>JH110. The ER gene composed of 849 nucleotides encodes a polypeptide with a calculated molecular mass of 31.4 kDa. The deduced amino acid sequence of ER showed a high degree of similarity to other members of the aldo-keto reductase superfamily including three ER isozymes from <it>Trichosporonoides megachiliensis </it>SNG-42. The intact coding region of ER from <it>C. magnoliae </it>JH110 was cloned, functionally expressed in <it>Escherichia coli </it>using a combined approach of gene fusion and molecular chaperone co-expression, and subsequently purified to homogeneity. The enzyme displayed a temperature and pH optimum at 42°C and 5.5, respectively. Among various aldoses, the <it>C. magnoliae </it>JH110 ER showed high specific activity for reduction of erythrose to the corresponding alcohol, erythritol. To explore the molecular basis of the catalysis of erythrose reduction with NADPH, homology structural modeling was performed. The result suggested that NADPH binding partners are completely conserved in the <it>C. magnoliae </it>JH110 ER. Furthermore, NADPH interacts with the side chains Lys252, Thr255, and Arg258, which could account for the enzyme's absolute requirement of NADPH over NADH.</p> <p>Conclusions</p> <p>A novel ER enzyme and its corresponding gene were isolated from <it>C. magnoliae </it>JH110. The <it>C. magnoliae </it>JH110 ER with high activity and catalytic efficiency would be very useful for <it>in vitro </it>erythritol production and could be applied for the production of erythritol in other microorganisms, which do not produce erythritol.</p

Effect of a New Prokinetic Agent DA-9701 Formulated with Corydalis Tuber and Pharbitidis Semen on Cytochrome P450 and UDP-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

DA-9701 is a new botanical drug composed of the extracts of Corydalis tuber and Pharbitidis semen, and it is used as an oral therapy for the treatment of functional dyspepsia in Korea. The inhibitory potentials of DA-9701 and its component herbs, Corydalis tuber and Pharbitidis semen, on the activities of seven major human cytochrome P450 (CYP) enzymes and four UDP-glucuronosyltransferase (UGT) enzymes in human liver microsomes were investigated using liquid chromatography-tandem mass spectrometry. DA-9701 and Corydalis tuber extract slightly inhibited UGT1A1-mediated etoposide glucuronidation, with 50% inhibitory concentration (IC50) values of 188 and 290 μg/mL, respectively. DA-9701 inhibited CYP2D6-catalyzed bufuralol 1′-hydroxylation with an inhibition constant (Ki) value of 6.3 μg/mL in a noncompetitive manner. Corydalis tuber extract competitively inhibited CYP2D6-mediated bufuralol 1′-hydroxylation, with a Ki value of 3.7 μg/mL, whereas Pharbitidis semen extract showed no inhibition. The volume in which the dose could be diluted to generate an IC50 equivalent concentration (volume per dose index) value of DA-9701 for inhibition of CYP2D6 activity was 1.16 L/dose, indicating that DA-9701 may not be a potent CYP2D6 inhibitor. Further clinical studies are warranted to evaluate the in vivo extent of the observed in vitro interactions

Acori graminei rhizoma Ameliorated Ibotenic Acid-Induced Amnesia in Rats

In the present study, we investigated the effects of Acori graminei rhizoma (AGR) on learning and memory for the Morris water maze task and on the central cholinergic system of the rats with excitotoxic medial septum (MS) lesion. On the water maze test, the rats were trained to find a platform that was in a fixed position during 6 days and then they received a 60 s probe trial in which the platform was removed from the pool on the 7th day. Ibotenic lesioning of the MS impaired the performance on the maze test and it caused degeneration of choline acetyltransferase and acetylcholine esterase in the hippocampus, which are markers of the central cholinergic system. Daily administrations of AGR (100 mg kg−1, i.p.) for 21 consecutive days produced reversals of the ibotenic acid-induced deficit in learning and memory. These treatments also reduced the loss of cholinergic immunoreactivity in the hippocampus that was induced by ibotenic acid. These results demonstrated that AGR ameliorated learning and memory deficits through their effects on the central nervous system, and neuroprotection was partly evaluated through the effect of AGR on the cholinergic system. Our studies suggest that AGR can possibly be used as treatment for Alzheimer's disease

AKARI Detection of the Infrared-Bright Supernova Remnant B0104-72.3 in the Small Magellanic Cloud

We present a serendipitous detection of the infrared-bright supernova remnant (SNR) B0104-72.3 in the Small Magellanic Cloud by the Infrared Camera (IRC) onboard AKARI. An elongated, partially complete shell is detected in all four observed IRC bands covering 2.6-15 um. The infrared shell surrounds radio, optical, and X-ray emission associated with the SNR and is probably a radiative SNR shell. This is the first detection of a SNR shell in this near/mid-infrared waveband in the Small Magellanic Cloud. The IRC color indicates that the infrared emission might be from shocked H2 molecules with some possible contributions from ionic lines. We conclude that B0104-72.3 is a middle-aged SNR interacting with molecular clouds, similar to the Galactic SNR IC 443. Our results highlight the potential of AKARI IRC observations in studying SNRs, especially for diagnosing SNR shocks.Comment: 12 pages with 3 figures, accepted for publication in AKARI PASJ special issu