• Korean Journal of Medicinal Crop Science
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• v.13 no.5
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• pp.276-283
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• 2005

Ascorbic acid is a great antioxidant and helps protect the body against pollutants. GDP-mannose pyrophosphorylase (GMPase) is a key enzyme in manufacturing GDP-mannose, a glycosyl donor for ascorbate and cell wall biosynthesis as well as for protein glycosylation. In this study, we described molecular cloning of a full-length cDNA from Potato (Solanum tuberosum L. cv. Jasim), using tuber. The cDNA isolated encoded a GDP-mannose pyrophosphrylase. The nucleotide sequence of pGMPC showed about 95%, 89% and 80% homology with S. tuberosum (AF022716), N. tabacum (AB066279) and A. thaliana (AF076484) cDNAs clone known as GMPase, respectively. We detected the expression of GMPase using RT-PCR. The highest expression of GMPase was found in stems, and the largest amount of ascorbic acid was also presented in stems. In contrast, the leaf showed minimal level of GMPase transcript and ascorbic acid content. We propose that GMPase expression patterns were similar to the changes of ascorbic acid content in the leaves treated with diverse stresses.

• BMB Reports
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• v.42 no.8
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• pp.523-528
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• 2009

Phosphomannose isomerase (PMI) catalyzes the interconversion of fructose-6-phosphate and mannose-6-phosphate in the extracellular polysaccharide (EPS) synthesis pathway. The gene encoding PMI in Sphingomonas chungbukensis DJ77 was cloned and expressed in E. coli. The pmi gene is 1,410 nucleotides long and the deduced amino acid sequence shares high homology with other bifunctional proteins that possess both PMI and GDP-mannose pyrophosphorylase (GMP) activities. The sequence analysis of PMI revealed two domains with three conserved motifs: a GMP domain at the N-terminus and a PMI domain at the C-terminus. Enzyme assays using the PMI protein confirmed its bifunctional activity. Both activities required divalent metal ions such as $Co^{2+}$, $Ca^{2+}$, $Mg^{2+}$, $Ni^{2+}$ or $Zn^{2+}$. Of these ions, $Co^{2+}$ was found to be the most effective activator of PMI. GDP-D-mannose was found to inhibit the PMI activity, suggesting feedback regulation of this pathway.

• Journal of Microbiology and Biotechnology
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• v.16 no.6
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• pp.961-964
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• 2006

GDP-mannosyltransferase (GMT) is an enzyme responsible for the addition of a mannose to glucose ($\alpha$[1$\rightarrow$3]) during biosynthesis of the water-soluble branched polysaccharide acetan in Acefobacter species. In an effort to obtain a cellulose-overproducing bacterium, a mutant defective in GMT of Acetobacter xylinum KCCM 10100 was constructed by single crossover homologous recombination using part of the aceA gene encoding GMT amplified by polymerase chain reaction. The GMT-disrupted mutant produced 23% more cellulose, but 16% less water-soluble polysaccharide than those of the wild-type strain. Analysis of the sugar composition by gel permeation chromatography revealed that water-soluble polysaccharides produced by the GMT-defective mutant contained no mannose molecule.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.509-510
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• 2003

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.588-588
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• 2003

• Journal of Microbiology and Biotechnology
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• v.28 no.8
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• pp.1293-1298
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• 2018

Phosphomannomutase (ManB) converts mannose-6-phosphate (M-6-P) to mannose-1-phosphate (M-1-P), which is a key metabolic precursor for the production of GDP-D-mannose used for production of glycoconjugates and post-translational modification of proteins. The aim of this study was to express the manB gene from Escherichia coli in Lactococcus lactis subsp. cremoris NZ9000 and to characterize the encoded enzyme. The manB gene from E. coli K12, of 1,371 bp and encoding 457 amino acids (52 kDa), was cloned and overexpressed in L. lactis NZ9000 using the nisin-controlled expression system. The enzyme was purified by Ni-NTA column chromatography and exhibited a specific activity of 5.34 units/mg, significantly higher than that of other previously reported ManB enzymes. The pH and temperature optima were 8.0 and $50^{\circ}C$, respectively. Interestingly, the ManB used in this study had two substrate specificity for both mannose-1-phosphate and glucose-1-phosphate, and the specific activity for glucose-1-phosphate was 3.76 units/mg showing 70% relative activity to that of mannose-1-phosphate. This is the first study on heterologous expression and characterization of ManB in lactic acid bacteria. The ManB expression system constructed in this study canbe used to synthesize rare sugars or glycoconjugates.

• Microbiology and Biotechnology Letters
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• v.33 no.2
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• pp.84-89
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• 2005

Phosphomannomutase (PMM) is a key enzyme in prokaryotes and eukaryotes, which catalyzes the conversion of ${\alpha}$-D-mannose 6-phosphate to ${\alpha}$-D-mannose 1-phosphate. The latter is the substrate for the synthesis of GDP-mannose, which serves as the mannosyl donor for many metabolic pathways in the cells. We report here on the isolation of a gene from a genomic library of Sphingomonas chungbukensis DJ77, the pmmC gene encoding phosphomannomutase. The gene was cloned into E. coli expression vector, and the sequence was analyzed. The ribosomal binding site GGAAG lays 5 bp upstream of the ORF of 750 bp, which is initiated by ATG codon and terminated by TAG. The predicted sequence of the enzyme consists of 249 amino acids with a molecular mass of 27.4 kDa and showed $86.9\%$ similarity to that of eukaryotic phosphomannomutase after bioinformatical analyses with the conserved domain search of NCBI. The purified gene product revealed the activity of phosphomannomutase. In conclusion, we confirmed that pmmC gene encodes phosphomannomutase actually.

• Parasites, Hosts and Diseases
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• v.44 no.1 s.137
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• pp.15-20
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• 2006

Free-living amoebae of the genus Acanthamoeba are causative agents of granulomatous amebic encephalitis and amebic keratitis. Because the virulence of Acanthamoeba culbertsoni cultured in the laboratory is restored by consecutive brain passages, we examined the genes induced in mouse brain-passaged A. culbertsoni by differential display reverse transcriptase polymerase chain reaction (DDRT-PCR). Enhanced A. culbertsoni virulence was observed during the second mouse brain passage, i.e., infected mouse mortality increased from $5\%\;to\;70\%.$ Ten cDNAs induced during mouse brain passage were identified by DDRT-PCR and this was confirmed by northern blot analysis. BlastX searches of these cDNAs indicated the upregulations of genes encoding predictive NADH-dehydrogenase, proteasomal ATPase, and GDP-mannose pyrophosphorylase B, which have previously been reported to be associated with A. culbertsoni virulence factors.

• Journal of Microbiology
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• v.45 no.1
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• pp.69-74
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• 2007

Escherichia coli is a clonal species, and occurs as both commensal and pathogenic strains, which are normally classified on the basis of their O, H, and K antigens. The O-antigen (O-specific polysaccharide), which consists of a series of oligosaccharide (O-unit) repeats, contributes major antigenic variability to the cell surface. The O-antigen gene cluster of E. coli O66 was sequenced in this study. The genes putatively responsible for the biosynthesis of dTDP-6-deoxy-L-talose and GDP-mannose, as well as those responsible for the transfer of sugars and for O-unit processing were identified based on their homology. The function of the wzy gene was confirmed by the results of a mutation test. Genes specific for E. coli O66 were identified via PCR screening against representatives of 186 E. coli and Shigella O type strains. The comparison of intergenic sequences located between galF and the O-antigen gene cluster in a range of E. coli and Shigella showed that this region may perform an important function in the homologous recombination of the O-antigen gene clusters.

• Journal of Microbiology and Biotechnology
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• v.22 no.10
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• pp.1324-1329
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• 2012

Phosphomannomutase (ManB) is involved in the biosynthesis of GDP-mannose, which is vital for numerous processes such as synthesis of carbohydrates, production of alginates and ascorbic acid, and post-translational modification of proteins. Here, we discovered that a deletion mutant of manB (BG101) in Streptomyces coelicolor (S. coelicolor) showed higher sensitivity to bacteriostatic chloramphenicol (CM) than the wild-type strain (M145), along with decreased production of CM metabolites. Deletion of manB also decreased the mRNA expression level of drug efflux pumps (i.e., cmlR1 and cmlR2) in S. coelicolor, resulting in increased sensitivity to CM. This is the first report on changes in antibiotic sensitivity to CM by deletion of one glycolysis-related enzyme in S. coelicolor, and the results suggest different approaches for studying the antibiotic-resistant mechanism and its regulation.