• Proceedings of the Korean Society for Bioinformatics Conference
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• 2003.10a
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• pp.277-287
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• 2003

Acetolactate synthase (ALS, EC 4.1.3.18; also referred to as acetohydroxy acid synthase) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine in microorganisms and plants. Recently X-ray structure of yeast ALS was available. Pair-wise alignment of yeast and tobacco ALS sequences revealed 63% sequence similarity. Using Deep View and automatic modeling on Swiss model server, we have generated reliable models of tobacco ALS based on yeast ALS template with a calculated pair-wise RMSD of 0.86 Angstrom. Functional roles of four residues located on the subunit interface (H142, El43, M350, and R376) were examined by site-directed mutagenesis. Seven mutants were generated and purified, of which three mutants (H142T, M350V, and R376F) were found to be inactivated under various assay conditions. The H142k mutant showed moderately altered kinetic properties. The E143A mutant increased 10-fold in K$_m$ value while other parameters remained unchanged. The M350C mutant was strongly resistant to three tested herbicides, while the R376k mutant can bind with herbicide carder at similar affinity to that of wild type enzyme, as determined by tryptophan quenching study. Except M350V mutant, all other mutants were ate to bind with cofactor FAD. Taken together, it is likely that residues H142 and E143 are located at the active site, while residues M350 and R376 are possibly located at the overlapping region of active site and herbicide binding site of the enzyme. Our data also allows us to hypothesize that the interaction between side chains of residues M350 and R376 are probably essential for the correct conformation of the active site. It remains to be elucidated that, whether the herbicide, upon binding with enzyme, inactivates the enzyme by causing change in the active site allosterically, which is unfavorable for catalytic activity.

• Molecules and Cells
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• v.39 no.3
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• pp.211-216
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• 2016

CYP107W1 from Streptomyces avermitilis is a cytochrome P450 enzyme involved in the biosynthesis of macrolide oligomycin A. A previous study reported that CYP107W1 regioselectively hydroxylated C12 of oligomycin C to produce oligomycin A, and the crystal structure of ligand free CYP107W1 was determined. Here, we analyzed the structural properties of the CYP107W1-oligomycin A complex and characterized the functional role of the Trp178 residue in CYP107W1. The crystal structure of the CYP107W1 complex with oligomycin A was determined at a resolution of $2.6{\AA}$. Oligomycin A is bound in the substrate access channel on the upper side of the prosthetic heme mainly by hydrophobic interactions. In particular, the Trp178 residue in the active site intercalates into the large macrolide ring, thereby guiding the substrate into the correct binding orientation for a productive P450 reaction. A Trp178 to Gly mutation resulted in the distortion of binding titration spectra with oligomycin A, whereas binding spectra with azoles were not affected. The Gly178 mutant's catalytic turnover number for the 12-hydroxylation reaction of oligomycin C was highly reduced. These results indicate that Trp178, located in the open pocket of the active site, may be a critical residue for the productive binding conformation of large macrolide substrates.

• Microbiology and Biotechnology Letters
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• v.13 no.2
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• pp.119-127
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• 1985

A series of Escherichia coli mutants were exmined for ability to convert glucose and ammonium salts into phenylalanine. This enabled the biochemical changes having major. effects on phenylaianine yield, and interactions between mutations, to be identified. Changes to the common pathway of aromatic biosynthesis having a major effects include desensitization of the first enzyme (3-deoxy-D-arabinoheptulosonate synthase) to end-product inhibition, and removal of repression of enzyme synthesis. It is suggested that the 3-deoxy-D-arabino-heptulosonate synthase Phe isoenzyme has a more important effect on yield. Similarly, removal of repression and end-product inhibition on the phenylalanine terminal pathway increased yield, and changes to both common and branch pathways were synergistic. Blockage of the typrosine and tryptophan pathways had minor effects on phenylalanine yield, and a mutation affecting aramatic amino acid transport (aroP) decreased yield. With multiple-mutation strains hish specific rates of product formation (ie 0.1-0.17g phenylalanine/g cells/h) were obtained.

• Bulletin of the Korean Chemical Society
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• v.27 no.4
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• pp.549-555
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• 2006

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6 also referred to as acetolactate synthase) catalyzes the first common step in the metabolic pathway leading to biosynthesis of the branched-chain amino acids in plants and microorganisms. Due to its presence in plants, AHAS is a target for the herbicides (sulfonylurea and imidazolinone), which act as potent inhibitors of the enzyme. Recently, we have shown [J. Kim, D.G. Baek, Y.T. Kim, J.D. Choi, M.Y. Yoon, Biochem. J. (2004) 384, 59-68] that the residues in the “mobile loop” 567-582 on the C-termini are involved in the binding/stabilization of the active dimer and ThDP (thiamin diphosphate) binding. In this study, we have demonstrated the role of the W573 in the mobile loop of the C-termini of tobacco AHAS. The substitution of this W573 residue caused significant perturbations in the activation process and in the binding site of ThDP. Position W573 plays a structurally important role in the binding of FAD, maintaining the enzyme active site in the required geometry for catalysis to occur. In here we propose that the tryptophan at position 573 is important for the catalytic process.

• Animal Bioscience
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• v.36 no.2
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• pp.229-237
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• 2023

Objective: This study evaluated the effects of dandelion supplements on lactation performance, circulating antioxidative activity and plasma metabolomics in primiparous dairy cows. Methods: A total of 60 mid-lactation dairy cows (milk yield = 34.29±0.34 kg/d; days in milk = 151.72±2.36 days) were divided into 4 treatment groups randomly, comprising the addition of dandelion at 0, 100, 200, 400 g/d per head. The experiment lasted for 8 weeks with an extra 10 days' pre-feeding period. Milk and blood samples were collected, and plasma samples were selected to perform metabolomics analysis. Results: Supplementing 200 g/d of dandelion increased the yield of milk and lactose (p≤0.05). The milk somatic cell counts (p≤0.05) were lower in all dandelion groups than those in the control group. The activity of glutathione peroxidase (p≤0.05) and superoxide dismutase (p≤0.05) were increased and plasma malondialdehyde (p = 0.01) was decreased when cows were fed 200 g/d dandelion. Plasma metabolomics analysis showed that 23 hub differential metabolites were identified in the 200 g/d dandelion group. These metabolites such as ribose, glutamic acid, valine, and phenylalanine were enriched in D-glutamine and D-glutamate metabolism (p = 0.06, impact value = 1), phenylalanine, tyrosine, and tryptophan biosynthesis (p = 0.05, impact value = 0.5), and starch and sucrose metabolism (p = 0.21, impact value = 0.13). Moreover, correlation analysis showed that circulating ribose, mannose, and glutamic acid were positively related to milk yield. Conclusion: Dandelion supplementation could improve lactation performance and elevate the plasma carbohydrate and amino acids metabolism and antioxidative activity. Supplementation of 200 g/d dandelion is recommended for lactating dairy cows.

• Journal of the Korean Chemical Society
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• v.42 no.3
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• pp.306-314
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• 1998

Acetolactate synthase (ALS) is the common enzyme in the biosynthesis of branched chain amino acids, Val, Leu, and Ile in bacteria, yeast, and higher plants. The enzyme is target site of several classes of structually diverse herbicides, including the sulfonylureas, the imidazolinones, the triazolopyrimidines, and the primidyl-oxy-benzoates. We have synthesized new triazolopyrimidine (TP) derivatives, and determined their inhibitory activities on barley ALS. $lC_{50}$ values for the active compounds were 3.2 nM-0.62 mM, and some of them appeared to be potent inhibitors. The progress curves for inhibition of ALS by TP4, a representative derivative, indicated that the extent of inhibition increased with incubation time. The inhibition of ALS by TP4 showed mixed-type inhibition with respect to pyruvate. Dual inhibition analyses of TP4 versus imidazolinone Cadre and feedback inhibitor Leu suggested that three different classes of inhibitors bind to ALS in a mutually exclusive manner. Chemical modification of tyrosyl residues of ALS decreased sensitivity of ALS to TP4, while modification of tryptophan and cysteine did not affect the sensitivity.

• Journal of Aquaculture
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• v.15 no.3
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• pp.157-163
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• 2002

Thirteen groups, each consisting of 25 juveniles (0.64 g) of the Korean rockfish, Sebastes schlegeli were reared in aquaria after period of one week conditioning. Each group was fed with one of the 13 diets at the rate of 7~10% body weight (on a dry-matter basis) 2 times a day for 6 weeks. Each diet was supplemented with one of the following amino acids at 3g/kg diet: Alanine (Ala), Arginine (Arg), Glycine(Gly), Glutamate (Glu), Histidine (His), Isoleusine(Ile), Lysine (Lys), Methionine (Met), Phenylalanine (Phe), Proline (Pro), Threonine (Thr), Tryptophan (Trp) or Valine (Val). Groups fed with Pro, Thr, Met or Gly-supplemented diet showed significantly (p<0.05) higher weight gain and faster specific growth rate than the groups fed on other diets, while those fed Pro, Thr, Met or Gly were not significantly different each other(P>0.05). Feed efficiency (FE) of fish fed Pro was significantly higher than those fed the other diets except that fed Thr (P<0.05). However, there was no significant difference between FE of fish fed Pro and Thr, and among FE of fish fed Thr, Met and Gly (P>0.05). The requirement of rockfish is higher for Met and Thr. Extra Pro and/or Gly may also stimulate biosynthesis of the nucleic acids (IMP, GMP) which are known as the feed stimulant in fish.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.12
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• pp.1784-1795
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• 2017

Objective: The study examined the effect of intravenous administration of bacterial endotoxin-lipopolysaccharide (LPS) -on the nocturnal secretion of melatonin and on the expression of enzymes of the melatonin biosynthetic pathway in the pineal gland of ewes, taking into account two different photoperiodic conditions: short-night (SN; n = 12) and long-night (LN; n = 12). Methods: In both experiments, animals (n = 12) were randomly divided into two groups: control (n = 6) and LPS-treated (n = 6) one. Two hours after sunset, animals received an injection of LPS or saline. Blood samples were collected starting one hour after sunset and continuing for 3 hours after the treatment. The ewes were euthanized 3 hours after LPS/saline treatment. The concentration of hormones in plasma was assayed by radioimmunoassay. In the pineal gland, the content of serotonin and its metabolite was determined by HPLC; whereas the expression of examined genes and protein was assayed using real-time polymerase chain reaction and Western Blot, respectively. Results: Endotoxin administration lowered (p<0.05) levels of circulating melatonin in animals from LN photoperiod only during the first hour after treatment, while in ewes from SN photoperiod only in the third hour after the injection. Inflammation more substantially suppressed biosynthesis of melatonin in ewes from SN photoperiod, which were also characterised by lower (p<0.05) cortisol concentrations after LPS treatment compared with animals from LN photoperiod. In the pineal gland of ewes subjected to SN photoperiod, LPS reduced (p<0.05) serotonin content and the expression of melatonin biosynthetic pathway enzymes, such as tryptophan hydroxylase and arylalkylamine-N-acetyltransferase. Pineal activity may be disturbed by circulating LPS and proinflammatory cytokines because the expression of mRNAs encoding their corresponding receptors was determined in this gland. Conclusion: The present study showed that peripheral inflammation reduces the secretion of melatonin, but this effect may be influenced by the photoperiod.

• Allergy, Asthma & Immunology Research
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• v.10 no.6
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• pp.628-647
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• 2018

Purpose: Obesity is associated with metabolic dysregulation, but the underlying metabolic signatures involving clinical and inflammatory profiles of obese asthma are largely unexplored. We aimed at identifying the metabolic signatures of obese asthma. Methods: Eligible subjects with obese (n = 11) and lean (n = 22) asthma underwent body composition and clinical assessment, sputum induction, and blood sampling. Sputum supernatant was assessed for interleukin $(IL)-1{\beta}$, -4, -5, -6, -13, and tumor necrosis factor $(TNF)-{\alpha}$, and serum was detected for leptin, adiponectin and C-reactive protein. Untargeted gas chromatography time-of-flight mass spectrometry (GC-TOF-MS)-based metabolic profiles in sputum, serum and peripheral blood monocular cells (PBMCs) were analyzed by orthogonal projections to latent structures-discriminate analysis (OPLS-DA) and pathway topology enrichment analysis. The differential metabolites were further validated by correlation analysis with body composition, and clinical and inflammatory profiles. Results: Body composition, asthma control, and the levels of $IL-1{\beta}$, -4, -13, leptin and adiponectin in obese asthmatics were significantly different from those in lean asthmatics. OPLS-DA analysis revealed 28 differential metabolites that distinguished obese from lean asthmatic subjects. The validation analysis identified 18 potential metabolic signatures (11 in sputum, 4 in serum and 2 in PBMCs) of obese asthmatics. Pathway topology enrichment analysis revealed that cyanoamino acid metabolism, caffeine metabolism, alanine, aspartate and glutamate metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, pentose phosphate pathway in sputum, and glyoxylate and dicarboxylate metabolism, glycerolipid metabolism and pentose phosphate pathway in serum are suggested to be significant pathways related to obese asthma. Conclusions: GC-TOF-MS-based metabolomics indicates obese asthma is characterized by a metabolic profile different from lean asthma. The potential metabolic signatures indicated novel immune-metabolic mechanisms in obese asthma with providing more phenotypic and therapeutic implications, which needs further replication and validation.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.51-51
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• 2001

Cocaine and amphetamine-regulated transcript (CART) is a satiety factor that is regulated by leptin. It was reported that the mice intracerebroventricularly injected with CART showed behavioral changes resembled with the typical behavioral alterations found in the mice carrying disorders in the brain serotonergic (5-HT) system. Hence, this study was conducted to find out the relationships between CART and 5-HT. We first examined the mRNA levels of CART after the injections of para-chlorophenylalanine (pCPA, 300 mg/kg i.p., single injection or daily for three consecutive days) in the rat brains by in situ hybridization using the mouse CART cDNA probe cloned in our laboratory. Systemic administrations of pCPA, a potent inhibitor of tryptophan hydroxylase, the rate limiting enzyme of 5-HT biosynthesis, acutely depletes the brain 5-HT transporter (5-HTT) in the dorsal raphe nucleus (DRN), which reuptakes terminal 5-HT. Results indicated that the mRNA level of CART significantly decreased in the arcuate nucleus, paraventricular nucleus, and lateral hypothalamic nucleus by three days of daily injection with pCPA with no noticeable change detected 24 hrs after the single injection. The message levels of 5-HTT in DRN decreased in both single and three days of injections. Secondly, to investigate whether CART affect to 5-HT, mouse genomic CART gene, which is consist of 3 exons and 2 introns and mouse neurofilament light (NF-L) chain promoter were cloned. Then, we constructed neuron specific expression vector, which was transfected into HeLa cell using lipid-mediated transfection system. Expression of GFP and CART linked to NF-L-chain promoter in the transfected HeLa cell were detected by using fluorescent microscope and RT-PCR. These results confirmed normal expression of DNA constructs in vitro. Then, to increase brain specific expression of CART in vivo transgenic mice carrying CART gene controlled the deleted NF-L-chain promoter were generated by the DNA microinjection into pronuclei of fertilized embryos. Transgenic mice were detected by Southern blot. Further study is necessary to examine CART expression and 5-HTT in these transgenic mice. Therefore, these results suggest that there maybe a positive molecular correlation between CART and 5-HT in responding to the stimuli.