• 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.

• Korean Journal of Environmental Agriculture
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• v.17 no.3
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• pp.239-245
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• 1998

To investigate the formation of bound residue with soil organic materials by oxidative coupling, nitroaromatics and their reduced metabolites, the insecticide parathion and the herbicide asulam were incubated with oxidoreductase, laccase or horseradish peroxidase, in the presence or absence of humic monomers. Most of aminotoluenes and amino-nitrophenols were completely transformed while most of nitrotoluenes and nitrophenols remained unchanged by a lactase or horseradish peroxidase in the presence or absence of humic monomers. Amino-nitrotoluenes were not transformed without humic monomers, but the addition of various humic monomers caused a considerable difference in the transformation of amino-nitrotoluenes by a lactase or horseradish peroxidase. Amino-nitrotoluenes were most transformed in the presence of catechol, syringaldehyde and protocatechuic acid. The insecticide parathion with nitro group and its metabolite were not mostly transformed in the presence or absence of humic monomers. The herbicide asulam with amino group remained unchanged without humic monomers as well, but the stimulating effect on the transformation of asulam was caused by the addition of catechol, syringaldehyde, protocatechuic acid or caffeic acid with a lactase.

• Journal of Life Science
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• v.17 no.2 s.82
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• pp.204-210
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• 2007

Previous reports indicated that the carboxyl terminal residues, glutamine276-threonine277 in particular, were important for actin affinity of the unacetylated smooth ${\alpha}-tropomyosin$. To determine the role of the glutamine and threonine residues in C-terminal region in actin binding, we constructed mutant striated muscle ${\alpha}-tropomyosin$ (TMs), in which these two residues were individually substituted. These mutant tropomyosins, designated TM18 (HT) and TM19 (QA), were overexpressed in E. coli as an either unacetylated form or Ala-Ser. (AS) dipeptide fusion form, and were analyzed F-actin affinity by cosedimentation. Unacetylated TM19 (QA) bound to actin approximately three times stronger than TM18 (HT) and much stronger than ST (HA). AS/TM19 (QA) showed four times stronger, in actin affinity than AS/ST (HA) while AS/TM14 (QT) bound to actin stronger to some extent than AS/TM18 (HT). These results suggested that the presence of Gln residue at 276 be primarily attributed to higher actin affinity of smooth ${\alpha}-tropomyosin$.

• Journal of Life Science
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• v.14 no.5
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• pp.770-777
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• 2004

It has been previously reported that unacetylated a-tropomyosin(TM) produced in E. coli failed to bind to actin while acetylated muscle TM and Ala-Ser dipeptide fusion TM (AS-TM) bound well to actin. In order to determine the structural requirement of the amino terminus for high actin affinity, a recombinant tropomyosin (Ala-TM) that a single Ala residue was added to the amino terminus of Ala-TM was constructed, overexpressed, and purified from E. coli. Actin affinity of Ala-TM was 2.3$\times$$10^{6}$$M^{-1}$, whereas that of unacetylated TM was considerably lower than 0.1$\times$$10^{-6}$$M^{-1}$ indicating that addition of a single Ala residue to the amino terminus drastically increased, at least twenty times, actin affinity of TM. Ala-TM, however, bound to actin about three times weaker than acetylated TM and AS- TM, implying that the addition of an Ala residue was insufficient for complete restoration of high actin affinity. While Ala-TM, AS-TM, and muscle TM showed inhibition and activation of actomyosin Sl ATPase activity depending on myosin Sl concentration, the degree of inhibition and activation was different from each other. AS-TM exhibited the greatest inhibition of the ATPase at low Sl concentration, whereas the greatest activation of the ATPase was observed with muscle TM. These results, together with previous findings, strongly suggested that local structure of the amino terminus is the crucial functional determinant of TM.

• Bulletin of the Korean Chemical Society
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• v.31 no.9
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• pp.2497-2502
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• 2010

Arg13 is a conserved active-site residue in all known Pi class glutathione S-transferases (GSTs) and in most Alpha class GSTs. To evaluate its contribution to substrate binding and catalysis of this residue, three mutants (R13A, R13K, and R13L) were expressed in Escherichia coli and purified by GSH affinity chromatography. The substitutions of Arg13 significantly affected GSH-conjugation activity, while scarcely affecting glutathione peroxidase or steroid isomerase activities. Mutation of Arg13 into Ala largely reduced the GSH-conjugation activity by approximately 85 - 95%, whereas substitutions by Lys and Leu barely affected activity. These results suggest that, in the GSH-conjugation activity of hGST P1-1, the contribution of Arg13 toward catalytic activity is highly dependent on substrate specificities and the size of the side chain at position 13. From the kinetic parameters, introduction of larger side chains at position 13 results in stronger affinity (Leu > Lys, Arg > Ala) towards GSH. The substitutions of Arg13 with alanine and leucine significantly affected $k_{cat}$, whereas substitution with Lys was similar to that of the wild type, indicating the significance of a positively charged residue at position 13. From the plots of log ($k_{cat}/{K_m}^{CDNB}$) against pH, the $pK_a$ values of the thiol group of GSH bound in R13A, R13K, and R13L were estimated to be 1.8, 1.4, and 1.8 pK units higher than the $pK_a$ value of the wild-type enzyme, demonstrating the contribution of the Arg13 guanidinium group to the electrostatic field in the active site. From these results, we suggest that contribution of Arg13 in substrate binding is highly dependent on the nature of the electrophilic substrates, while in the catalytic mechanism, it stabilizes the GSH thiolate through hydrogen bonding.

• The Korean Journal of Pesticide Science
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• v.3 no.1
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• pp.66-77
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• 1999

In order to elucidate the fate of the residues of the bipyridylium herbicide paraquat in soil, maize plants were grown for 4 weeks on the specially-made pots filled with two different types of soils containing fresh and 6-week-aged residues of [$^{14}C$]paraquat, respectively. The mineralization of [$^{14}C$]paraquat to $^{14}CO_{2}$ during the aging period and the cultivation period of maize plants amounted to $0.13{\sim}0.18%$ and $0.02{\sim}0.17%$, respectively, of the original $^{14}C$ activities. At harvest the roots and shoots contained less than 0.1% and 0.01% of the originally applied $^{14}C$ activities, respectively, whereas the $^{14}C$ activities remaining in soil were more than 97% in both soils. The water extractability of the soil where maize plants were grown for 4 weeks was less than 1.2% of the original $^{14}C$ activities. Most of the non-extractable soil-bound residues of [$^{14}C$]paraquat were incorporated into the humin fraction. Soil pHs during the aging of soil B and after cultivation in all treatments increased. The distribution of the $^{14}C$ activities in subcellular particles of the maize plant roots was the highest in the residue fraction(incompletely homogenized tissue). Dehydrogenase activities increased after vegetation, regardless of soil aging.

• The Korean Journal of Mycology
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• v.17 no.4
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• pp.223-228
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• 1989

This study was performed to obtain higher yield rate of protein-bound polysac-charide extracted from cultured broth and fruit body of Coriolus versieolor, basidiomycetes. 16002 strain produced 0.58%, the highest yield rate among four strains, and there was no significantly different yield in other three strains. The optimal liquid media were CVT-III to obtain higher yield of protein-bound polysaccharide comparing with the various culture media. Compared with several materials for substitution of medium source, the residue extract media of Coriolus versieolor fruit-body were yield rate similar to control, and addition of the extracts of ginseng residues increased 11.4-48%. And also compared with fruit bodies that were artificially cultured on the oak longs, 16002 strain produced the highest yield similar to the result of cultured broth. Fruit body of the prematures stage was more effective to obtain high yield rate.

• Journal of Photoscience
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• v.9 no.2
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• pp.302-304
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• 2002

pharaonis phoborhodopsin (ppR), a photophobic sensor of haloalkaliphilic bacteria, Natronobacterium phar-aonis, has retinal as a chromophore covalently bound to Lys in G-helix via a protonated Schiff base (PSB), as is the same as bacteriorhodopsin (bR). For ppR, the corresponding counter-ion is Asp residue (Asp75) located in C-helix. Here we investigated the influence of the protonated state of this counter-ion and its location on the chromophore configuration. Under alkaline condition, the chromophore configuration of D75E mutant was analyzed by HPLC. D75E had a much larger content of 13-cis isomer: the ratio of 13-cis to all-trans was 6:4 while the wild-type had this ratio of 1 :9. On the other hand, under acidic condition where Glu was associated, D75E had no 13-cis retinal isomer. Mutants whose Asp75 was replaced by neutral amino acids (D75N and D75Q) did not contain 13-cis retinal. Furthermore, retinal isomer compositions and the change in the visible ab- sorption spectra (indicating the dissociation state of Glu75) were measured under varying pH, and these were almost the same dependencies. These results indicate that an important factor determining the 13-cis isomer content is the presence of negative charge of the counter-ion against PSB, but not the size of this residue. Com- parison between the wild-type and D75E in alkaline solutions indicates the influence of the location of the counter-ion.

• Korean Journal of Environmental Agriculture
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• v.13 no.3
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• pp.262-270
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• 1994

The established methods in the residue analysis of endosulfan require an extensive sample clean-up prior to quantification by relatively complex equipment. A radioimmunoassay(RIA) provides a simple procedure with theoretically higher sensitivity and specificity necessitating only a minimum of sample clean-up. Endosulfan-specific antibodies were developed in rabbits by using a bovine serum albumin(BSA) conjugate wherein the alcohol form of endosulfan was multiply bound to the protein via succinylation. Produced antibodies showed the high titers to endosulfan-BSA(1 : 32,000). An RIA method was developed in water and carp by using $^{14}C-labeled$ endosulfan as a tracer. The lowest detection amount of endosulfan was 1 ng in the liver, kidneys, gut and water samples, and 3 ng in the whole body sample of carp without any clean-up, corresponding to 0.1 ppb of endosulfan.

• BMB Reports
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• v.37 no.3
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• pp.330-338
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• 2004

Different types of cardiotoxin (I-V and n) were isolated and purified from the venom of the Taiwan cobra (Naja naja atra). The effects of these cardiotoxins were studied on membrane-bound acetylcholinesterase, which was isolated from a sheep's brain cortex. The results showed that cardiotoxins I-III, V, and n activated the enzyme by modification of substrate inhibition, but cardiotoxin IV's reaction was different. The inhibition and activation of acetylcholinesterase were linked to the functions of the hydrophobicity index, presence of a cationic cluster, and the accessible arginine residue. Our results indicate that Cardiotoxins have neither a cationic cluster nor an arginine residue in their surface area of loop I; therefore, in contrast to fasciculin, cardiotoxins are attached by loop II to the peripheral site of the enzyme. As a result, fasciculin seems to stabilize nonfunctional conformation, but cardiotoxins seem to stabilize the functional conformation of the enzyme. Based on our experimental and theoretical findings, similar secondary and tertiary structures of cardiotoxins and fasciculin seem to have an opposite function once they interact with acetylcholinesterase.