• Korean Journal of Microbiology
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• v.38 no.3
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• pp.173-179
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• 2002

The functions of riboflavin synthesis genes ( ribI,II,III and IV) found immediately downstream of luxG in the lux operon from Photobacterium species were identified using the biochemical and genetical analysis. The ribI-III gene codes for protein corresponding to that coded by the second (riboflavin synthase), third (3,4-dihydroxy 2-butanone 4-phosphate synthase/GTP cyclohydrolase II) and fourth (lumazine synthase) gene, respectively, of Bacillus subtilis rib operon with the respective gene procuct sharing 41-50% amino acid sequence identity. Unexpectedly, the sequence of the ribIV product of Photobacterium phosphoreum does not correspond in sequence to the protein encoded by the fifth rib gene of Bacillus subtilis. Instead the gene (ribIV) codes for a polypeptide similar in sequence to GTP cyclohydrolase II of Escherichia coli and the carboxy terminal domain of the third rib gene from Bacillus subtilis. Complementation of Escherichia coli riboflavin auxotrophs showed that the function of the gene products of ribII and ribIV are DHBP synthase and GTP cyclohydrolase II, respectively. In addition the experiment, showing that increase in thermal stability of riboflavin synthase coded by ribIon coexpression with ribIII, provided indirect evidence that the latter gene codes for lumazine synthase.

• Korean Journal of Microbiology
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• v.47 no.1
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• pp.14-21
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• 2011

Riboflavin synthase catalyzes the formation of one molecule of each riboflavin and 5-amino-6-ribitylamino-2,4-pyrimidinedione by the transfer of a 4-carbon moiety between two molecules of the substrates, 6,7-dimetyl-8-ribityllumazine. The most remarkable feature is the sequence similarity between the N-terminal half (1-97) and the C-terminal half domain (99-213). To investigate the structure and fluorescent characteristics of the N-terminal half of riboflavin synthase (N-RS) in Escherichia coli, more than 10 mutant genes coding for the mutated N-terminal domain of riboflavin synthase were generated by polymerase chain reaction. The genes coding for the proteins were inserted into pQE vector designed for easy purification of protein by 6X-His tagging system, expressed, and the proteins were purified. Almost all mutated N-terminal domain of riboflavin synthases bind to 6,7-dimethyl-8-ribityllumazine and riboflavin as fluorescent ligands. However, N-RS C47D and N-RS ET66,67DQ mutant proteins show colorless, indicating that fluorescent ligands were dissociated during purification. In addition, most mutated proteins show low fluorescent intensity comparing to N-RS wild type, whereas N-RS C48S posses stronger fluorescent intensity than that of wild type protein. Based on this result, N-RS C48S can be used as the tool for high throughput screening system for searching for the compound with inhibitory effect for the riboflavin synthase.

• BMB Reports
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• v.40 no.2
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• pp.239-246
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• 2007

Riboflavin synthase from Escherichia coli is a homotrimer of 23.4 kDa subunits and catalyzes the formation of one molecule each of riboflavin and 5-amino-6-ribitylamino- 2,4(1H,3H)-pyrimidinedione by the transfer of a 4-carbon moiety between two molecules of the substrate, 6,7- dimethyl-8-ribityllumazine. Each subunit comprises two closely similar folding domains. Recombinant expression of the N-terminal domain is known to provide a $C_2$-symmetric homodimer. In this study, the binding properties of wild type as well as two mutated proteins of N-terminal domain of riboflavin synthase with various ligands were tested. The replacement of the amino acid residue A43, located in the second shell of riboflavin synthase active center, in the recombinant N-terminal domain dimer reduces the affinity for 6,7-dimethyl-8-ribityllumazine. The mutation of the amino acid residue C48 forming part of activity cavity of the enzyme causes significant $^{19}F$ NMR chemical shift modulation of trifluoromethyl derivatives of 6,7-dimethyl-8-ribityllumazine in complex with the protein, while substitution of A43 results in smaller chemical shift changes.

• Korean Journal of Microbiology
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• v.41 no.4
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• pp.306-311
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• 2005

In this study, the amino-terminal half truncated lump and the whole lump genes from Photobacterium phosphoreum coding for the lumazine protein were cloned by polymerase chain reaction and expressed in Escherichia coli. To identifiy of the binding site of the ligand or substrate, the amino acid identities from the sequences of the lumazine protein, yellow fluorescent protein, and riboflavin synthase from different organisms were also compared and analyzed.

• Journal of the Korean Chemical Society
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• v.65 no.6
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• pp.489-494
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• 2021

• Korean Journal of Microbiology
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• v.36 no.1
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• pp.1-7
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• 2000

The genes involved in riboflavin synthesis (ribI, II, III, and IV) were found immediately downstream of luxG in the lux operon from Photobacterium species. The single stranded DNA containing the intergenic region of lux genes and rib genes from Photobacterium phosphoreum was fully protected by P. phosphoreum mRNA from the S1 nuclease mapping assay suggesting that a transcriptional terminator was not present in the region. In addition, the levels of riboflavin synthase activity in P. phosphoreum was increased during the development of bacterial bioluminescence in the same fashion as the luciferase and fatty acid reductase activities. Insertion of the Photobacterium leiognathi DNA extending from luxB to ribII, between a strong lux promoter and a reporter gene (chloramphenicol acetyltransferase, CAT) and transferred by conjugation into P. leiognathi, did not affect expression of reporter gene. Moreover the CAT gene was not expressed in an analogous construct missing the lux promoter indicating that a promoter was not present in this region. Based on the data here, it can be concluded that the lux genes and rib genes in Photobacterium species are under common regulation.

• Bulletin of the Korean Chemical Society
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• v.31 no.4
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• pp.1017-1020
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• 2010

• Bulletin of the Korean Chemical Society
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• v.34 no.6
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• pp.1673-1678
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• 2013

Lumazine protein is a member of the riboflavin synthase superfamily and the intense fluorescence is caused by non-covalently bound to 6,7-dimethyl 8-ribityllumazine. To figure out the binding modes and the structure of the N-terminal domain of lumazine protein, the wild type of protein extending to amino acid 118 (N-LumP 118 Wt) and mutants of N-LumP 118 V41W, S48W, T50W, D64W, and A66W from Photobacterium leiognathi were purified. The biochemical properties of the wild type and mutants of N-LumP 118 proteins were analyzed by absorbance and fluorescence spectroscope. The peak of absorbance and fluorescence of lumazine ligand were shifted to longer wavelength on binding to N-LumPs. The observed absorbance value at 410 nm of lumazine bound to N-LumP 118 proteins indicate that one mole of N-LumP 118 proteins bind to one mole of ligand of lumazine. Fluorescence analysis show that the maximum peak of fluorescence of N-LumP S48W was shifted to the longest wavelength by binding with 6,7-dimethyl 8-ribityllumazine and was shown to the greatest quench effect by acrylamide among all tryptophan mutants.

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.24 no.1
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• pp.78-85
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• 2011

Background and Objectives : The aim of this study was to investigate anti-inflammatory and anti-oxidant effects of Hyunggaeyungyo-tang(HYT) on RAW 264.7 cells. Material and Methods : Two types of experiments were implemented for this study: first, the experiment to study the anti-oxidant effect of HYT using riboflavin; second, in vitro experiment to investigate the suppression of NF-${\kappa}$B activation using RAW 264.7 cells (I${\kappa}$B kinase and induce nitric oxide synthase mRNA expression) Results : 1. The anti-oxidant effects of HYT was dose-dependantly increased. 2. The RAW 264.7cells were treated with LPS for 1 hours prior to the addition of indicated concentrations(0.4,-1.0mg/$m\ell$) of HYT, and the cells were further incubated for 24 hours. The LPS-induced IKK & iNOS mRNA expression were dose-dependantly decreased in HYT treated RAW 264.7cells. Conclusion : The results suggest that HYT is significantly effective in the treatment of inflammation through the suppression of NF-${\kappa}$B activation and iNOS production.

• Journal of Preventive Veterinary Medicine
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• v.42 no.4
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• pp.157-162
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• 2018

This study evaluated the protective effects of a combination of eight B. abortus recombinant proteins that were cloned and expressed into a pMal vector system and $DH5{\alpha}$: nucleoside diphosphate kinase (rNdk), 50S ribosomal protein (rL7/L12), malate dehydrogenase (rMDH), DNA starvation/stationary phase protection protein (rDps), elongation factor (rTsf), arginase (rRocF), superoxide dismutase (rSodC), and riboflavin synthase subunit beta (rRibH). The proteins were induced, purified, and administered intraperitoneally into BALB/c mice. The mice were immunized three times at weeks 0, 2, and 5 and then infected intraperitoneally (IP) with $5{\times}10^4CFU$ of virulent B. abortus 544 one week after the last immunization. The spleens were collected and the bacterial burden was evaluated at four weeks post-infection. The results showed that this combination produced a significant reduction of the bacterial burden in the spleen with a log reduction of 1.01 compared to the PBS group. Cytokine analysis revealed induction of the cell-mediated immune response in that TNF (tumor necrosis factor) and proinflammatory cytokines IL-6 (Interleukin 6) and MCP-1 (macrophage chemoattractant protein-1) were elevated significantly. In summary, vaccination with a combination of eight different proteins induced a significant protective effect indicative of a cell mediated immune response.