• BMB Reports
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• v.37 no.4
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• pp.503-506
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• 2004

Glucose-1-phosphate uridylyltransferase from E. coli K12 was used to convert uridine-5'-triphosphate and glucose-1-phosphate to UDP-D-glucose. The conversion was efficient and completed within 5 minutes under the employed conditions. In addition, thymidine-5'-monophosphate kinase and acetate kinase were proven to be non-specific, converting udridine-5'-monophosphate to uridine-5'-triphosphate with 55% conversion after 6 h, which was much slower than the production of TTP under the same conditions (complete conversion within one hour). Since these two reactions could proceed under the same conditions, a one-pot synthesis of UDP-D-glucose with ATP regeneration was designed from easily available starting materials, and conversion up to 40% by HPLC peak integration was achieved given a reaction time of 4 h.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.217-221
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• 2002

dTDP-D-glucose 4,6-dehydratase (TDPDH) catalyzes the conversion of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose, and requires $NAD^＋$ as a coenzyme for its catalytic activity. The dTDP-D-glucose 4,6-dehydratase from Streptomyces antibioticus $Tu{\ddot}99$ tightly binds $NAD^＋$ [19]. In order to determine the role of lysine-148 in the $NAD^＋$ binding, the lysine of the dTDP-D-glucose 4,6-dehydratase from Streptomyces antibioticus $Tu{\ddot}99$ was mutated to various amino acids by site-directed mutagenesis. The catalytic activity of the four mutated enzymes of TDPDH did not recover after addition of $NAD^＋$ . However, the activity of K159A, the mutated enzyme of UDP-D-glucose 4-epimerase (UDPE), recovered after the addition of $NAD^＋$ [15]. Although dTDP-glucose 4,6-dehydratase, and UDP-galactose (glucose) 4-epimerase are members of the short-chain dehydrogenase/reductase SDR family and the lysine-148 of TDPDH was highly conserved as in UDPE (Lys-159), the function of the lysine-148 of TDPDH was different from that of UDPE. The mutated enzymes showed that the lysine-148 of the dTDP-D-glucose 4,6-dehydratase played no role in the $NAD^＋$ binding. Accordingly, it is suggested that the lysine-148 of the dTDP-D-glucose 4,6-dehydratase is involved in the folding of TDPDH.

• BMB Reports
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• v.32 no.4
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• pp.358-362
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• 1999

dTDP-D-glucose 4,6-dehydratase as an oxidoreductase catalyzes the conversion of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose, which is essential for the formation of 6-deoxysugars. dTDP-D-glucose 4,6-dehydratase shows remarkable sterochemical convergence in which displacement of the C-6 hydroxyl group by a C-4 hydrogen proceeds intramolecularly with inversion of configuration. The reaction mechanism is known to be oxidation, dehydration, and reduction by bases mediating proton transfer and $NAD^+$ cofactor. In this study, the bases in the active site domain are proposed to be His-79 and His-300 from a comparison of the peptides of the dehydratase and UDP-D-glucose epimerase. His-79 and His-300 were mutated to prepare the mutants H79L (mutation of histidine to leucine at the 79th amino acid) and H300A (mutation of histidine to alanine at the 300th amino acid) by site-directed mutagenesis. The H79L protein was inactive, showing that His-79 participates in the reaction mechanism.

• Journal of Microbiology and Biotechnology
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• v.28 no.2
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• pp.298-304
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• 2018

The single-vessel multienzyme UDP-${\alpha}$-$\text\tiny{D}$-glucose recycling system was coupled with a forward glucosylation reaction to produce novel glucose moiety-conjugated derivatives of different tetracycline antibiotic analogs. Among five tetracycline analogs used for the reaction, four molecules (chlorotetracycline, doxytetracycline, meclotetracycline, and minotetracycline) were accepted by a glycosyltransferase enzyme, YjiC, from Bacillus licheniformis to produce glucoside derivatives. However, the enzyme was unable to conjugate sugar units to rolitetracycline. All glucosides of tetracycline derivatives were characterized by ultraviolet absorbance maxima, ultra-pressure liquid chromatography coupled with photodiode array, and high-resolution quadruple time-of-flight electrospray mass spectrometry analyses. These synthesized glucosides are novel tetracycline derivatives.

• 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 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.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.

• Molecules and Cells
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• v.28 no.4
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• pp.397-401
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• 2009

Flavonoids are a group of polyphenolic compounds that have been recognized as important due to their physiological and pharmacological roles and their health benefits. Glycosylation of flavonoids has a wide range of effects on flavonoid solubility, stability, and bioavailability. We previously generated the E. coli BL21 (DE3) ${\Delta}pgi$ host by deleting the glucose-phosphate isomerase (Pgi) gene in E. coli BL21 (DE3). This host was further engineered for whole-cell biotransformation by integration of galU from E. coli K12, and expression of calS8 (UDP-glucose dehydrogenase) and calS9 (UDP-glucuronic acid decarboxylase) from Micromonospora echinospora spp. calichensis and arGt-4 (7-O-glycosyltransferase) from Arabidopsis thaliana to form E. coli (US89Gt-4), which is expected to produce glycosylated flavonoids. To test the designed system, the engineered host was fed with naringenin as a substrate, and naringenin 7-O-xyloside, a glycosylated naringenin product, was detected. Product was verified by HPLC-LC/MS and ESI-MS/MS analyses. The reconstructed host can be applied for the production of various classes of glycosylated flavonoids.

• 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 $CuSO_4$.

• Molecules and Cells
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• v.22 no.2
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• pp.233-238
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• 2006

Endogenous salicylic acid (SA) and its predominant conjugates, SA 2-O-${\beta}$-D-glucoside (SAG) and the glucose ester of SA (SGE), increase dramatically during plant defense responses. Here I report the isolation and characterization of an Arabidopsis thaliana UDP-glucose:SA glucosyltransferase1 (AtSGT1) gene using a tobacco SGT gene previously reported, whose product catalyzes the formation of both SAG and SGE. The recombinant AtSGT1 protein had significant activities with SA and benzoic acid, and synthesized SAG and SGE. Northern blot analysis showed that AtSGT1 was rapidly induced both by exogenous SA and infection with the bacterial pathogen Pseudomonas syringae, indicating that pathogen-inducible AtSGT1 expression is an early disease response and may be involved in the accumulation of glucosyl SA during pathogenesis.