Jung-Do Choi

Characterization of acetohydroxyacid synthase from Mycobacterium tuberculosis and the identification of its new inhibitor from the screening of a chemical library

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate‐ (ThDP‐) and FAD‐dependent enzyme that catalyzes the first common step in the biosynthetic pathway of the branched‐amino acids such as leucine, isoleucine, and valine. The genes of AHAS from Mycobacterium tuberculosis were cloned, and overexpressed in E. coli and purified to homogeneity. The purified AHAS from M. tuberculosis is effectively inhibited by pyrazosulfuron ethyl (PSE), an inhibitor of plant AHAS enzyme, with the IC50 (inhibitory concentration 50%) of 0.87 μM. The kinetic parameters of M. tuberculosis AHAS were determined, and an enzyme activity assay system using 96‐well microplate was designed. After screening of a chemical library composed of 5600 compounds using the assay system, a new class of AHAS inhibitor was identified with the IC50 in the range of 1.8–2.6 μM. One of the identified compounds (KHG20612) further showed growth inhibition activity against various strains of M. tuberculosis. The correlation of the inhibitory activity of the identified compound against AHAS to the cell growth inhibition activity suggested that AHAS might be served as a target protein for the development of novel anti‐tuberculosis therapeutics.

The Effects of Areca Catechu L Extract on Anti‐Inflammation and Anti‐Melanogenesis

Ethanolic extract (CC‐516) from Areca catechu L was prepared and its various biological activities were evaluated, CC‐516 showed potent anti‐oxidative, free radical scavenging, and anti‐hyaluronidase activity. Anti‐oxidative effect of CC‐516 (IC : 45.4 μg/ml) was lower than butylated hydroxytoluene (IC : 5μg ml), but similar to tocopherol and higher than ascorbic acid. Especially, CC‐516 exhibited relatively high free radical scavenging activity (IC : 10.2 μg/ml) compared to control. CC‐516 inhibited effectively hyaluronidase activity (IC : 416 μg/ml), showed inhibition in vivo on delayed hypersensitivity as well as croton‐oil induced ear edema in mice when it was topically applied. These results strongly suggest that CC‐516 may reduce immunoregulatory/inflammatory skin trouble. Also, from the results, we have elucidated that CC‐516 showed anti‐allergic and anti‐cytotoxicity activity. The whitening effect of CC‐516 shown by the inhibition of mushroom tyrosinase activity with IC of 0.48 mg/ml and of melanin synthesis in B16 melanoma cells. This study indicates that CC‐516 is effective on anti‐inflammatory/anti‐melanogenesis, and can be used as a new agent for cosmetics.

The effects of areca catechu L extract on anti-aging.

The anti‐aging effects of Areca catechu L extract (CC‐516) on skin were investigate both in vitro and in vivo. The CC‐516 has a high proportion of proline (13%) of free amino acid content. The inhibitory effect of CC‐516 on the elastase exhibited 37 to 90% inhibition by 10 to 250 μg/ml concentration; the IC values with 40.8 μg/ml for porcine pancreatic elastase (PPE) and 48.1 μg/ml for human leukocyte elastase (HLE), respectively. One of the effects of elastase is that it is known to reduce the number of elastin fibers at the level of the enzyme deposition. The number of elastin fibers was increased when we drift from the deposit number of elastase with 100 mg/ml of CC‐516. CC‐516 showed protection of elastic fiber against degradation by the enzyme in an ex vivo assay. The CC‐516 increased proliferation of human fibroblast cell by 85% at 10 concentration, compared with control, whereas the increase by ascorbic acid was 50%. The collagen synthesis was increased by 40% at 10 % concentration of CC‐516. The treatment with CC‐516 improved skin hydration, the skin elasticity, and skin wrinkles. From this study, we suggest that CC‐516 can be used as a new anti‐aging component for cosmetics.

Roles of lysine 219 and 255 residues in tobacco acetolactate synthase

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine. The ALS is the target of several classes of herbicides, including the sulfonylureas, the imidazolinones, and the triazolopyrimidines. The roles of three well-conserved lysine residues (K219, K255, K299) in tobacco ALS were determined using site-directed mutagenesis. The mutation of K219Q inactivated the enzyme and abolished the binding affinity for cofactor FAD. However, the secondary structure of the enzyme was not changed significantly by the mutation. Both mutants, K255F and K255Q, showed strong resistance to three classes of herbicides Londax (a sulfonylurea), Cadre (an imidazolinone), and TP (a triazolopyrimidine). In addition, there was no difference in the secondary structures of wALS and K255F. On the other hand, the mutation of K299Q did not show any significant effect on the kinetic properties or any sensitivity to the herbicides. These results suggest that Lys219 is located at the active site and is likely involved in the binding of FAD, and that Lys255 is located at a binding site common for the three herbicides in tobacco ALS.

Amino acid residues conferring herbicide resistance in tobacco acetohydroxy acid synthase

The enzyme AHAS (acetohydroxy acid synthase), which is involved in the biosynthesis of valine, leucine and isoleucine, is the target of several classes of herbicides. A model of tobacco AHAS was generated based on the X-ray structure of yeast AHAS. Well conserved residues at the herbicide-binding site were identified, and the roles of three of these residues (Phe-205, Val-570 and Phe-577) were determined by site-directed mutagenesis. The Phe-205 mutants F205A, F205H, F205W and F205Y showed markedly decreased levels of catalytic efficiency, and cross-resistance to two or three classes of herbicides, i.e. Londax (a sulphonylurea herbicide), Cadre (an imidazolinone herbicide) and TP (a triazolopyrimidine derivative). None of the mutations caused significant changes in the secondary or tertiary structure of the enzyme. Four mutants of Phe-577, i.e. F577D, F577E, F577K and F577R, showed unaltered V(max) values, but substantially decreased catalytic efficiency. However, these mutants were highly resistant to two or three of the tested herbicides. The three mutants F577D, F577E and F577R had a similar secondary structure to that of wild-type AHAS. Conservative mutations of Phe-577, i.e. F577W and F577Y, did not affect the kinetic properties of the enzyme or its inhibition by herbicides. The mutation Val-570 to Asn abolished the binding affinity of the enzyme for FAD as well as its activity, and also caused a change in the tertiary structure of AHAS. However, the mutant V570Q was active, but resistant to two classes of herbicides, i.e. Londax and TP. The conservative mutant V570I was substantially reduced in catalytic efficiency and moderately resistant to the three herbicides. The results of this study suggest that residues Phe-205, Val-570 and Phe-577 in tobacco AHAS are located at or near the binding site that is common for the three classes of herbicides. In addition, Phe-205 and Val-570 are probably located at the herbicide-binding site that may overlap partially with the active site. Selected mutants of Phe-577 are expected to be utilized to construct herbicide-resistant transgenic plants.

Actinomycin D as a novel SH2 domain ligand inhibits Shc/Grb2 interaction in B104-1-1 (neu*-transformed NIH3T3) and SAA (hEGFR-overexpressed NIH3T3) cells

Actinomycins, a family of bicyclic chromopeptide lactones with strong antineoplastic activity, were screened as inhibitors of Shc/Grb2 interaction in in vitro assay systems. To investigate the effects of actinomycin D on Shc/Grb2 interaction in cell‐based experiments, we used SAA (normal hEGFR‐overexpressed NIH3T3) cells and B104‐1‐1 (neu*‐transformed NIH3T3) cells, because a large number of the Shc/Grb2 complexes were detected. Associated protein complexes containing Shc were immunoprecipitated from actinomycin D‐treated cell lysates with polyclonal anti‐Shc antibody. Then the association with Grb2 was assessed by immunoblotting with monoclonal anti‐Grb2 antibody. The result of the immunoblotting experiment revealed that actinomycin D inhibited Shc/Grb2 interaction in a dose‐dependent manner in both B104‐1‐1 and EGF‐stimulated SAA cells. The inhibition of Shc/Grb2 interaction by actinomycin D in B104‐1‐1 cells also reduced tyrosine phosphorylation of MAP kinase (Erk1/Erk2), one of the major components in the Ras‐MAP kinase signaling pathway. These results suggest that actinomycin D could be a non‐phosphorylated natural and cellular membrane‐permeable SH2 domain antagonist.

Roles of conserved methionine residues in tobacco acetolactate synthase.

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine. ALS is the target of several classes of herbicides, including the sulfonylureas, the imidazolinones, and the triazolopyrimidines. The conserved methionine residues of ALS from plants were identified by multiple sequence alignment using ClustalW. The alignment of 17 ALS sequences from plants revealed 149 identical residues, seven of which were methionine residues. The roles of three well-conserved methionine residues (M350, M512, and M569) in tobacco ALS were determined using site-directed mutagenesis. The mutation of M350V, M512V, and M569V inactivated the enzyme and abolished the binding affinity for cofactor FAD. Nevertheless, the secondary structure of each of the mutants determined by CD spectrum was not affected significantly by the mutation. Both M350C and M569C mutants were strongly resistant to three classes of herbicides, Londax (a sulfonylurea), Cadre (an imidazolinone), and TP (a triazolopyrimidine), while M512C mutant did not show a significant resistance to the herbicides. The mutant M350C was more sensitive to pH change, while the mutant M569C showed a profile for pH dependence activity similar to that of wild type. These results suggest that M512 residue is likely located at or near the active site, and that M350 and M569 residues are probably located at the overlapping region between the active site and a common herbicide binding site.

Caveat: mycoplasma arginine deiminase masquerading as nitric oxide synthase in cell cultures.

We used confluent cultures of dog gallbladder epithelial cells, stimulated by conditioned medium from a culture of human neonatal foreskin fibroblasts, to establish the presence of inducible nitric oxide synthase (NOS, EC 1.14.13.39). Assay was by conversion of radiolabeled arginine to citrulline. By 4 days after addition of the conditioned medium, a relatively high level of activity was observed. However, further study showed that the enzyme did not require addition of the usual cofactors for maximal activity (NADPH, FAD, FMN and tetrahydrobiopterin) and was stable in the absence of anti-proteolytic agents. Our suspicion that this enzyme might not be NOS but arginine deiminase (EC 3.5.3.6) was confirmed by enzyme purification and by the liberation of ammonia during enzyme reaction. This enzyme, which is absent from primates and virtually confined to single-cell organisms, suggested the presence of Mycoplasma, a common contaminant of cell cultures, and it was subsequently confirmed that the fibroblast culture was a source of Mycoplasma. With the widespread interest in nitric oxide and NOS, and common use of the convenient [3H]arginine assay, there is a considerable danger of the two enzymes being confused. At the very least, it is necessary to check for activity in the absence of added cofactors.

Cloning, characterization and evaluation of potent inhibitors of Shigella sonnei acetohydroxyacid synthase catalytic subunit.

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent plant and microbial enzyme that catalyzes the first common step in the biosynthesis of essential amino acids such as leucine, isoleucine and valine. To identify strong potent inhibitors against Shigella sonnei (S. sonnei) AHAS, we cloned and characterized the catalytic subunit of S. sonnei AHAS and found two potent chemicals (KHG20612, KHG25240) that inhibit 87-93% S. sonnei AHAS activity at an inhibitor concentration of 100uM. The purified S. sonnei AHAS had a size of 65kDa on SDS-PAGE. The enzyme kinetics revealed that the enzyme has a K(m) of 8.01mM and a specific activity of 0.117U/mg. The cofactor activation constant (K(s)) for ThDP and (K(c)) for Mg(++) were 0.01mM and 0.18mM, respectively. The dissociation constant (K(d)) for ThDP was found to be 0.14mM by tryptophan fluorescence quenching. The inhibition kinetics of inhibitor KHG20612 revealed an un-competitive inhibition mode with a K(ii) of 2.65mM and an IC(50) of 9.3μM, whereas KHG25240 was a non-competitive inhibitor with a K(ii of) 5.2mM, K(is) of 1.62mM and an IC(50) of 12.1μM. Based on the S. sonnei AHAS homology model structure, the docking of inhibitor KHG20612 is predicted to occur through hydrogen bonding with Met 257 at a 1.7Å distance with a low negative binding energy of -9.8kcal/mol. This current study provides an impetus for the development of a novel strong antibacterial agent targeting AHAS based on these potent inhibitor scaffolds.

5-Demethylovalicin, as a Methionine Aminopeptidase-2 Inhibitor Produced by Chrysosporium

5-Demethylovalicin was isolated from the fermentation broth Chrysosporium lucknowense and the structure was identified by spectroscopic methods. 5-Demethylovalicin inhibited the recombinant human MetAP-2 (IC(50)=17.7 nM) and the growth of human umbilical vein endothelial cells (HUVEC; IC(50)=100 nM) in cell proliferation assay without cytotoxicity on the transformed and cancer cell lines.