• BMB Reports
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• v.40 no.5
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• pp.690-696
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• 2007

There are conflicting views for the polymerization process of human UDP-glucose dehydrogenase (UGDH) and no clear evidence has been reported yet. Based on crystal coordinates for Streptococcus pyogenes UGDH, we made double mutant A222Q/S233G. The double mutagenesis had no effects on expression, stability, and secondary structure. Interestingly, A222Q/S233G was a dimeric form and showed an UGDH activity, although it showed increased $K_m$ values for substrates. These results suggest that Ala222 and Ser233 play an important role in maintaining the hexameric structure and the reduced binding affinities for substrates are attributable to its altered subunit communication although quaternary structure may not be critical for catalysis.

• Korean Journal of Food Science and Technology
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• v.45 no.2
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• pp.156-160
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• 2013

A duplex polymerase chain reaction (PCR) method was developed to detect unapproved genetically modified (GM) potato (EH92-527-1) in Korea. The UDP-glucose pyrophosphorylase (UGP) gene was selected as an endogenous reference gene for potato and used to validate the specificity for 14 different crops. The primer pair EH92-F/R was designed to amplify the junction sequence between the genome and transgenic region introduced in GM potato. Its specificity was also validated using several different GM events. The detection limit of the duplex PCR method is approximately 0.05%. This duplex PCR method could be useful for monitoring cultivation of unauthorized GM potato in Korea.

• Journal of Microbiology and Biotechnology
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• v.26 no.3
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• pp.477-482
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• 2016

A uridine diphosphate-glucose:sterol glycosyltransferase-encoding gene was isolated and cloned from the established fosmid library of Micromonospora rhodorangea ATCC 27932 that usually produces the aminoglycoside antibiotic geneticin. The gene consists of 1,185 base pairs and encodes a 41.4 kDa protein, which was heterologously expressed in Escherichia coli BL21(DE3). In silico analyses of the deduced gene product suggested that it is a member of the family 1 glycosyltransferases. The recombinant protein MrSGT was able to catalyze the transfer of a glucosyl moiety onto the C-3 hydroxy function in sterols (β-sitosterol, campesterol, and cholesterol), resulting in the corresponding steryl glucosides (β-sitosterol-3-O-β-ᴅ-glucoside, campesterol-3-O-β-ᴅ-glucoside, and cholesterol-3-O-β-ᴅ-glucoside). This enzyme prefers phytosterols to cholesterol, and also shows substrate flexibility to some extent, in that it could recognize a number of acceptor substrates.

• Journal of Life Science
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• v.20 no.2
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• pp.292-297
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• 2010

Anthocyanin, a phenolic compound, is a pigment that shows blue or red color in the fruit, petal and other tissues. It is an important factor in grape berry skin pigment and accumulates only in the skin. This skin-specific accumulation of anthocyanin has been reported to be regulated by the ufgt gene which encodes UDP-glucose: flavonoid 3-O-glucosyltransferase that participates in the biosynthesis of anthocyanin. The ufgt gene is expressed only in berry skin, while the other genes involved in the biosynthetic pathway are expressed in both skin and flesh tissues. In order to determine whether anthocyanin accumulation is primarily regulated by compartment of UFGT, a ufgt cDNA clone was isolated from grape berry, its open reading frame was ligated in pBI121 vector in either a sense or an antisense orientation under the control of the CaMV35S promoter and the recombinant constructs were incorporated into tobacco plants. Several transgenic lines were selected and characterized to determine the level of expression of the grapevine ufgt transcript and endogenous homologs of tobacco. Compared to the wild-type, the amount of anthocyanins in sense transgenic plants increased by 44%, while the amount of anthocyanins in antisense transgenic plants decreased by 88%. In addition, the color of flowers became intense in the sense transgenic plants. These results suggest that over-expression or repression of the ufgt gene affected the accumulation of anthocyanin in flowers of tobacco.

• Journal of Microbiology and Biotechnology
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• v.16 no.8
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• pp.1311-1315
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• 2006

Aminocyclitol antibiotic validamycin A, a prime control agent for sheath blight disease of rice plants, is biosynthesized by Streptomyces hygroscopicus var. limoneus. Within the validamycin biosynthetic gene cluster, vldBC forms an operon of vldABC with vidA, the gene encoding 2-epi-5-epi-valiolone synthase. Biochemical studies, employing the recombinant proteins from Escherichia coli, established VldB and VldC as D-glucose $\alpha$-1-phosphate uridylyltransferase and glucokinase, respectively. This finding substantiates that the validamycin biosynthetic gene cluster harbors genes encoding the enzymes for UDP-glucose formation from glucose. Therefore, we propose that validamycin biosynthesis employs its own catalysts to generate UDP-glucose, but not depending on the primary metabolism.

• Journal of Microbiology and Biotechnology
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• v.30 no.7
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• pp.1092-1096
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• 2020

YjiC, a glycosyltransferase from Bacillus licheniformis, is a well-known versatile enzyme for glycosylation of diverse substrates. Although a number of O-glycosylated products have been produced using YjiC, no report has been updated for nucleophilic N-, S-, and C- glycosylation. Here, we report the additional functional capacity of YjiC for nucleophilic N- and S- glycosylation using a broad substrate spectrum including UDP-α-D-glucose, UDP-N-acetyl glucosamine, UDP-N-acetylgalactosamine, UDP-α-D-glucuronic acid, TDP-α-L-rhamnose, TDP-α-D-viosamine, and GDP-α-L-fucose as donor and various amine and thiol groups containing natural products as acceptor substrates. The results revealed YjiC as a promiscuous enzyme for conjugating diverse sugars at amine and thiol functional groups of small molecules applicable for generating glycofunctionalized chemical diversity libraries. The glycosylated products were analyzed using HPLC and LC/MS and compared with previous reports.

• Journal of Microbiology and Biotechnology
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• v.26 no.11
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• pp.1918-1923
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• 2016

Two baicalein derivatives, baicalin and oroxylin A, were synthesized in this study. These derivatives exhibit diverse biological activities, such as anxiolytic and anticancer activities as well as memory enhancement. In order to synthesize baicalin from aglycon baicalein using Escherichia coli, we utilized a glycosyltransferase that regioselectively transfers glucuronic acid from UDP-glucuronic acid to the 7-hydroxy group of baicalein. To increase baicalin productivity, an araA deletion E. coli mutant, which accumulates UDP-glucuronic acid, was used, and ugd, which converts UDP-glucose to UDP-glucuronic acid, was overexpressed. Using these strategies, approximately $720.3{\mu}M$ baicalin was synthesized from $1,000{\mu}M$ baicalein. Oroxylin A was then synthesized from baicalein. Two O-methyltransferases (OMTs), ROMT-15 and POMT-9, were tested to examine the production of oroxylin A from baicalein. E. coli harboring ROMT-15 and E. coli harboring POMT-9 produced reaction products that had different retention times, indicating that they are methylated at different positions; the structure of the reaction product from POMT-9 was consistent with oroxylin A, whereas that from ROMT-15 was 7-O-methyl baicalein. Using E. coli harboring POMT-9, approximately 50.3 mg/l of oroxylin A ($177{\mu}M$) was synthesized from 54 mg/l baicalein ($200{\mu}M$).

• Molecules and Cells
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• v.37 no.2
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• pp.172-177
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• 2014

A glycosyltransferase, YjiC, from Bacillus licheniformis has been used for the modification of the commercially available isoflavonoids genistein, daidzein, biochanin A and formononetin. The in vitro glycosylation reaction, using UDP-${\alpha}$-D-glucose as a donor for the glucose moiety and aforementioned four acceptor molecules, showed the prominent glycosylation at 4' and 7 hydroxyl groups, but not at the $5^{th}$ hydroxyl group of the A-ring, resulting in the production of genistein 4'-O-${\beta}$-D-glucoside, genistein 7-O-${\beta}$-D-glucoside (genistin), genistein 4',7-O-${\beta}$-D-diglucoside, biochanin A-7-O-${\beta}$-D-glucoside (sissotrin), daidzein 4'-O-${\beta}$-D-glucoside, daidzein 7-O-${\beta}$-D-glucoside (daidzin), daidzein 4', 7-O-${\beta}$-D-diglucoside, and formononetin 7-O-${\beta}$-D-glucoside (ononin). The structures of all the products were elucidated using high performance liquid chromatography-photo diode array and high resolution quadrupole time-of-flight electrospray ionization mass spectrometry (HR QTOF-ESI/MS) analysis, and were compared with commercially available standard compounds. Significantly higher bioconversion rates of all four isoflavonoids was observed in both in vitro as well as in vivo bioconversion reactions. The in vivo fermentation of the isoflavonoids by applying engineered E. coli $BL21(DE3)/{\Delta}pgi{\Delta}zwf{\Delta}ushA$ overexpressing phosphoglucomutase (pgm) and glucose 1-phosphate uridyltransferase (galU), along with YjiC, found more than 60% average conversion of $200{\mu}M$ of supplemented isoflavonoids, without any additional UDP-${\alpha}$-D-glucose added in fermentation medium, which could be very beneficial to large scale industrial production of isoflavonoid glucosides.

• Journal of Applied Biological Chemistry
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• v.42 no.1
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• pp.12-18
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• 1999

Sucrose synthase (EC 2.4.1.13) has been purified from the plant cytosolic fraction of chickpea (Cicer arietinum L. cv. Amethyst) nodules. The native enzyme had a molecular mass of $356{\pm}15kD$. The subunit molecular mass was $87{\pm}2kD$, and a tetrameric structure is proposed for sucrose synthase of chickpea nodule. Optimum activities in the sucrose cleavage and synthesis directions were at pH 6.5 and 9.0, respectively. The purified enzyme displayed typical hyperbolic kinetics with substrates in cleavage and synthesis reactions. Chickpea nodules sucrose synthase had a high affinity for UDP ($K_m$, $8.0{\mu}M$) and relatively low affinities for ADP ($K_m$, 0.23 mM), CDP ($K_m$, 0.87 mM), and GDP ($K_m$, 1.51 mM). The $K_m$ for sucrose was 29.4 mM. In the synthesis reaction, UDP-glucose ($K_m$, $24.1{\mu}M$) was a more effective glucosyl donor than ADP-glucose ($K_m$, 2.7 mM), and the $K_m$ for fructose was 5.4 mM. Divalent cations, such as $Ca^{2+}$, $Mg^{2+}$, and $Mn^{2+}$, stimulated the enzyme activity in both the cleavage and synthesis directions, and the enzyme was very sensitive to inhibition by $HgCl_2$ and $CuSO_4$.

• Journal of Microbiology
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• v.43 no.2
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• pp.191-195
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• 2005

In this study, we attempted to characterize the Synechococcus sp. pee 7942 mutant resultant from a disruption in the gene encoding UDP-glucose: tetrahydrobiopterin a-glucosyltransferase (BGluT). 2D­PAGE followed by MALDI-TOF mass spectrometry revealed that phycocyanin rod linker protein 33K was one of the proteins expressed at lower level in the BGluT mutant. BGluT mutant cells were also determined to be more sensitive to high light stress. This is because photosynthetic O$_2$ exchange rates were significantly decreased, due to the reduced number of functional PSIs relative to the wild type cells. These results suggested that, in Synechococcus sp. pee 7942, BH4-glucoside might be involved in photosynthetic photoprotection.